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<!DOCTYPE html> <html lang="en" class="grade-c"> <head> <title>Identification and characterization of a bacterial core methionine synthase | Scientific Reports</title> <link rel="alternate" type="application/rss+xml" href="https://www.nature.com/srep.rss"/> <link rel="preconnect" href="https://cmp.nature.com" crossorigin> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="applicable-device" content="pc,mobile"> <meta name="viewport" content="width=device-width,initial-scale=1.0,maximum-scale=5,user-scalable=yes"> <meta name="360-site-verification" content="5a2dc4ab3fcb9b0393241ffbbb490480" /> <script data-test="dataLayer"> window.dataLayer = [{"content":{"category":{"contentType":"article","legacy":{"webtrendsPrimaryArticleType":"research","webtrendsSubjectTerms":"enzymes;methylases;molecular-evolution;transferases","webtrendsContentCategory":null,"webtrendsContentCollection":null,"webtrendsContentGroup":"Scientific 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Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetECBDB in E. coli and purified the 38 kDa protein. Core-MetECBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (KM ≈ 240 µM) and L-homocysteine (KM ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a kcat ≈ 60 s−1. Core-MetECBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetECBDB and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain.","datePublished":"2020-02-07T00:00:00Z","dateModified":"2020-02-07T00:00:00Z","pageStart":"1","pageEnd":"13","license":"http://creativecommons.org/licenses/by/4.0/","sameAs":"https://doi.org/10.1038/s41598-020-58873-z","keywords":["Enzymes","Methylases","Molecular evolution","Transferases","Science","Humanities and Social 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Rev. Nutr.; citation_title=Metabolism of sulfur-containing amino acids; citation_author=MH Stipanuk; citation_volume=6; citation_publication_date=1986; citation_pages=179-209; citation_doi=10.1146/annurev.nu.06.070186.001143; citation_id=CR2"/> <meta name="citation_reference" content="citation_journal_title=J. Inherit. Metab. Dis.; citation_title=Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur; citation_author=MH Stipanuk, I Ueki; citation_volume=34; citation_publication_date=2011; citation_pages=17-32; citation_doi=10.1007/s10545-009-9006-9; citation_id=CR3"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. Chem.; citation_title=S-adenosylmethionine; a new intermediate formed enzymatically from L-methionine and adenosinetriphosphate; citation_author=GL Cantoni; citation_volume=204; citation_publication_date=1953; citation_pages=403-416; citation_id=CR4"/> <meta name="citation_reference" content="citation_journal_title=Gene; citation_title=Evolutionary plasticity of methionine biosynthesis; citation_author=U Gophna, E Bapteste, WF Doolittle, D Biran, EZ Ron; citation_volume=355; citation_publication_date=2005; citation_pages=48-57; citation_doi=10.1016/j.gene.2005.05.028; citation_id=CR5"/> <meta name="citation_reference" content="citation_journal_title=Biochm Soc. Trans.; citation_title=S-adenosylmethionine. A ‘poor man’s coenzyme B12’ in the reaction of lysine 2,3-aminomutase; citation_author=PA Frey, MD Ballinger, GH Reed; citation_volume=26; citation_publication_date=1998; citation_pages=304-310; citation_doi=10.1042/bst0260304; citation_id=CR6"/> <meta name="citation_reference" content="citation_journal_title=Biochem.; citation_title=Two enzymic mechanisms for the methylation of homocysteine by extracts of Escherichia coli; citation_author=MA Foster, G Tejerina, JR Guest, DD Woods; citation_volume=92; citation_publication_date=1964; citation_pages=476-488; citation_id=CR7"/> <meta name="citation_reference" content="citation_journal_title=Mol. Microbiol.; citation_title=Regulation of methionine synthesis in Escherichia coli; citation_author=H Weissbach, N Brot; citation_volume=5; citation_publication_date=1991; citation_pages=1593-1597; citation_doi=10.1111/j.1365-2958.1991.tb01905.x; citation_id=CR8"/> <meta name="citation_reference" content="citation_journal_title=FEBS J.; citation_title=Crystal structure and solution characterization of the activation domain of human methionine synthase; citation_author=KR Wolthers; citation_volume=274; citation_publication_date=2007; citation_pages=738-750; citation_doi=10.1111/j.1742-4658.2006.05618.x; citation_id=CR9"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. 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Cell; citation_title=S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase; citation_author=F Bourgis; citation_volume=11; citation_publication_date=1999; citation_pages=1485-1497; citation_doi=10.1105/tpc.11.8.1485; citation_id=CR12"/> <meta name="citation_reference" content="citation_journal_title=Curr. Opin. Chem. Biol.; citation_title=Enzyme-catalyzed methyl transfers to thiols: the role of zinc; citation_author=RG Matthews, CW Goulding; citation_volume=1; citation_publication_date=1997; citation_pages=332-339; citation_doi=10.1016/S1367-5931(97)80070-1; citation_id=CR13"/> <meta name="citation_reference" content="citation_journal_title=Proc. Natl Acad. Sci. USA; citation_title=A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor; citation_author=S Datta, M Koutmos, KA Pattridge, ML Ludwig, RG Matthews; citation_volume=105; citation_publication_date=2008; citation_pages=4115-4120; citation_doi=10.1073/pnas.0800329105; citation_id=CR14"/> <meta name="citation_reference" content="citation_journal_title=Biochem.; citation_title=Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli; citation_author=JT Drummond, S Huang, RM Blumenthal, RG Matthews; citation_volume=32; citation_publication_date=1993; citation_pages=9290-9295; citation_doi=10.1021/bi00087a005; citation_id=CR15"/> <meta name="citation_reference" content="citation_journal_title=Biochem.; citation_title=Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine; citation_author=CW Goulding, D Postigo, RG Matthews; citation_volume=36; citation_publication_date=1997; citation_pages=8082-8091; citation_doi=10.1021/bi9705164; citation_id=CR16"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. Chem.; citation_title=Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate; citation_author=J-L Ferrer, S Ravanel, M Robert, R Dumas; citation_volume=279; citation_publication_date=2004; citation_pages=44235-44238; citation_doi=10.1074/jbc.C400325200; citation_id=CR17"/> <meta name="citation_reference" content="citation_journal_title=J. Mol. Biol.; citation_title=Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans. A dynamic zinc-inversion model; citation_author=T-M Fu; citation_volume=412; citation_publication_date=2011; citation_pages=688-697; citation_doi=10.1016/j.jmb.2011.08.005; citation_id=CR18"/> <meta name="citation_reference" content="citation_journal_title=PLoS Biol.; citation_title=Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication; citation_author=R Pejchal, ML Ludwig; citation_volume=3; citation_publication_date=2005; citation_pages=254-265; citation_doi=10.1371/journal.pbio.0030254; citation_id=CR19"/> <meta name="citation_reference" content="citation_journal_title=Nat.; citation_title=Bacterial dehalorespiration with chlorinated benzenes; citation_author=L Adrian, U Szewzyk, J Wecke, H Görisch; citation_volume=408; citation_publication_date=2000; citation_pages=580-583; citation_doi=10.1038/35046063; citation_id=CR20"/> <meta name="citation_reference" content="citation_journal_title=Appl. Env. Microbiol.; citation_title=Reductive dehalogenation of chlorobenzene congeners in cell extracts of Dehalococcoides sp. strain CBDB1; citation_author=T Hölscher, H Gorisch, L Adrian; citation_volume=69; citation_publication_date=2003; citation_pages=2999-3001; citation_doi=10.1128/AEM.69.5.2999-3001.2003; citation_id=CR21"/> <meta name="citation_reference" content="citation_journal_title=Int. J. Syst. Evol. Microbiol.; citation_title=Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi; citation_author=FE Löffler; citation_volume=63; citation_publication_date=2013; citation_pages=625-635; citation_doi=10.1099/ijs.0.034926-0; citation_id=CR22"/> <meta name="citation_reference" content="citation_journal_title=BMC genomics; citation_title=Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization; citation_author=Y Zhang, DA Rodionov, MS Gelfand, VN Gladyshev; citation_volume=10; citation_publication_date=2009; citation_doi=10.1186/1471-2164-10-78; citation_id=CR23"/> <meta name="citation_reference" content="citation_journal_title=Appl. Env. Microbiol.; citation_title=Identification of a chlorobenzene reductive dehalogenase in Dehalococcoides sp. strain CBDB1; citation_author=L Adrian, J Rahnenführer, J Gobom, T Hölscher; citation_volume=73; citation_publication_date=2007; citation_pages=7717-7724; citation_doi=10.1128/AEM.01649-07; citation_id=CR24"/> <meta name="citation_reference" content="citation_journal_title=Philos. Trans. R. Soc. Lond.; citation_title=Organic cofactors in the metabolism of Dehalococcoides mccartyi strains; citation_author=CJ Schipp, E Marco-Urrea, A Kublik, J Seifert, L Adrian; citation_volume=368; citation_publication_date=2013; citation_pages=20120321; citation_doi=10.1098/rstb.2012.0321; citation_id=CR25"/> <meta name="citation_reference" content="citation_journal_title=Nat. Biotechnol.; citation_title=Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1; citation_author=M Kube; citation_volume=23; citation_publication_date=2005; citation_pages=1269-1273; citation_doi=10.1038/nbt1131; citation_id=CR26"/> <meta name="citation_reference" content="citation_journal_title=Sci.; citation_title=Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes; citation_author=R Seshadri; citation_volume=307; citation_publication_date=2005; citation_pages=105-108; citation_doi=10.1126/science.1102226; citation_id=CR27"/> <meta name="citation_reference" content="citation_journal_title=Appl. Env. Microbiol.; citation_title=Versatility in corrinoid salvaging and remodeling pathways supports corrinoid-dependent metabolism in Dehalococcoides mccartyi; citation_author=S Yi; citation_volume=78; citation_publication_date=2012; citation_pages=7745-7752; citation_doi=10.1128/AEM.02150-12; citation_id=CR28"/> <meta name="citation_reference" content="citation_journal_title=Appl. Env. Microbiol.; citation_title=Unexpected specificity of interspecies cobamide transfer from Geobacter spp. to organohalide-respiring Dehalococcoides mccartyi strains; citation_author=J Yan, KM Ritalahti, DD Wagner, FE Löffler; citation_volume=78; citation_publication_date=2012; citation_pages=6630-6636; citation_doi=10.1128/AEM.01535-12; citation_id=CR29"/> <meta name="citation_reference" content="citation_journal_title=J. Bacteriol.; citation_title=Investigation of carbon metabolism in “Dehalococcoides ethenogenes” strain 195 by use of isotopomer and transcriptomic analyses; citation_author=YJ Tang; citation_volume=191; citation_publication_date=2009; citation_pages=5224-5231; citation_doi=10.1128/JB.00085-09; citation_id=CR30"/> <meta name="citation_reference" content="citation_journal_title=J. Bacteriol.; citation_title=Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1; citation_author=E Marco-Urrea, J Seifert, Mvon Bergen, L Adrian; citation_volume=194; citation_publication_date=2012; citation_pages=4169-4177; citation_doi=10.1128/JB.00049-12; citation_id=CR31"/> <meta name="citation_reference" content="citation_journal_title=Proc. Natl Acad. Sci. USA; citation_title=Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi; citation_author=W-Q Zhuang; citation_volume=111; citation_publication_date=2014; citation_pages=6419-6424; citation_doi=10.1073/pnas.1321542111; citation_id=CR32"/> <meta name="citation_reference" content="citation_journal_title=Eur. J. Biochem.; citation_title=Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum; citation_author=I Schröder, RK Thauer; citation_volume=263; citation_publication_date=1999; citation_pages=789-796; citation_doi=10.1046/j.1432-1327.1999.00559.x; citation_id=CR33"/> <meta name="citation_reference" content="citation_journal_title=Nucleic Acids Res.; citation_title=KEGG: kyoto encyclopedia of genes and genomes; citation_author=M Kanehisa, S Goto; citation_volume=28; citation_publication_date=2000; citation_pages=27-30; citation_doi=10.1093/nar/28.1.27; citation_id=CR34"/> <meta name="citation_reference" content="citation_journal_title=Nucleic Acids Res.; citation_title=KEGG as a reference resource for gene and protein annotation; citation_author=M Kanehisa, Y Sato, M Kawashima, M Furumichi, M Tanabe; citation_volume=44; citation_publication_date=2016; citation_pages=457-462; citation_doi=10.1093/nar/gkv1070; citation_id=CR35"/> <meta name="citation_reference" content="citation_journal_title=Nucleic Acids Res.; citation_title=KEGG: new perspectives on genomes, pathways, diseases and drugs; citation_author=M Kanehisa, M Furumichi, M Tanabe, Y Sato, K Morishima; citation_volume=45; citation_publication_date=2017; citation_pages=353-361; citation_doi=10.1093/nar/gkw1092; citation_id=CR36"/> <meta name="citation_reference" content="citation_journal_title=Arch. Biochem. Biophys.; citation_title=Fungal cobalamin-independent methionine synthase: Insights from the model organism, Neurospora crassa; citation_author=RW Wheatley, KKS Ng, M Kapoor; citation_volume=590; citation_publication_date=2016; citation_pages=125-137; citation_doi=10.1016/j.abb.2015.11.037; citation_id=CR37"/> <meta name="citation_reference" content="citation_journal_title=Methods Enzymol.; citation_title=Measurement of energetics of conformational change in cobalamin-dependent methionine synthase; citation_author=V Bandarian, RG Matthews; citation_volume=380; citation_publication_date=2004; citation_pages=152-169; citation_doi=10.1016/S0076-6879(04)80007-7; citation_id=CR38"/> <meta name="citation_reference" content="citation_journal_title=Photochem. Photobiol.; citation_title=Photodegradation of 5-methyltetrahydrofolate: biophysical aspects; citation_author=AH Steindal, A Juzeniene, A Johnsson, J Moan; citation_volume=82; citation_publication_date=2006; citation_pages=1651-1655; citation_doi=10.1111/j.1751-1097.2006.tb09826.x; citation_id=CR39"/> <meta name="citation_reference" content="citation_journal_title=J. Agric. Food Chem.; citation_title=Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments; citation_author=PHCJ Verlinde; citation_volume=58; citation_publication_date=2010; citation_pages=6190-6199; citation_doi=10.1021/jf9041134; citation_id=CR40"/> <meta name="citation_reference" content="citation_journal_title=J. Food Sci.; citation_title=Thermal oxidation studies on reduced folate, L-5-methyltetrahydrofolic acid (L-5-MTHF) and strategies for stabilization using food matrices; citation_author=Y Liu; citation_volume=77; citation_publication_date=2012; citation_pages=C236-43; citation_doi=10.1111/j.1750-3841.2011.02561.x; citation_id=CR41"/> <meta name="citation_reference" content="citation_journal_title=Proc. Natl Acad. Sci. USA; citation_title=Primordial synthesis of amines and amino acids in a 1958 Miller H2S-rich spark discharge experiment; citation_author=ET Parker; citation_volume=108; citation_publication_date=2011; citation_pages=5526-5531; citation_doi=10.1073/pnas.1019191108; citation_id=CR42"/> <meta name="citation_reference" content="citation_journal_title=Orig. Life Evol. Biosph.; citation_title=Prebiotic synthesis of methionine and other sulfur-containing organic compounds on the primitive Earth: a contemporary reassessment based on an unpublished 1958 Stanley Miller experiment; citation_author=ET Parker; citation_volume=41; citation_publication_date=2011; citation_pages=201-212; citation_doi=10.1007/s11084-010-9228-8; citation_id=CR43"/> <meta name="citation_reference" content="citation_journal_title=Angew. Chem. Int. Ed.; citation_title=Spontaneous emergence of S-adenosylmethionine and the evolution of methylation; citation_author=P Laurino, DS Tawfik; citation_volume=56; citation_publication_date=2017; citation_pages=343-345; citation_doi=10.1002/anie.201609615; citation_id=CR44"/> <meta name="citation_reference" content="citation_journal_title=Angew. Chem. Int. Ed.; citation_title=Energy production in anaerobic organisms; citation_author=K Decker, K Jungermann, RK Thauer; citation_volume=9; citation_publication_date=1970; citation_pages=138-158; citation_doi=10.1002/anie.197001381; citation_id=CR45"/> <meta name="citation_reference" content="citation_journal_title=Cold Spring Harb. Perspect. Biol.; citation_title=Early microbial evolution: the age of anaerobes; citation_author=WF Martin, FL Sousa; citation_volume=8; citation_publication_date=2015; citation_pages=a018127; citation_doi=10.1101/cshperspect.a018127; citation_id=CR46"/> <meta name="citation_reference" content="citation_journal_title=J. Mol. Evol.; citation_title=Ribonucleotide reduction and the possible role of cobalamin in evolution; citation_author=SR Dickman; citation_volume=10; citation_publication_date=1977; citation_pages=251-260; citation_doi=10.1007/BF01764600; citation_id=CR47"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. Chem.; citation_title=The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein; citation_author=S Menon, SW Ragsdale; citation_volume=274; citation_publication_date=1999; citation_pages=11513-11518; citation_doi=10.1074/jbc.274.17.11513; citation_id=CR48"/> <meta name="citation_reference" content="citation_journal_title=Microbiol.; citation_title=Biochemistry of methanogenesis: a tribute to Marjory Stephenson; citation_author=RK Thauer; citation_volume=144; citation_publication_date=1998; citation_pages=2377-2406; citation_doi=10.1099/00221287-144-9-2377; citation_id=CR49"/> <meta name="citation_reference" content="citation_journal_title=Biochem.; citation_title=Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme; citation_author=JC González, K Peariso, JE Penner-Hahn, RG Matthews; citation_volume=35; citation_publication_date=1996; citation_pages=12228-12234; citation_doi=10.1021/bi9615452; citation_id=CR50"/> <meta name="citation_reference" content="citation_journal_title=Biochem.; citation_title=Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme; citation_author=V Frasca, RV Banerjee, WR Dunham, RH Sands, RG Matthews; citation_volume=27; citation_publication_date=1988; citation_pages=8458-8465; citation_doi=10.1021/bi00422a025; citation_id=CR51"/> <meta name="citation_reference" content="citation_journal_title=Helvetica Chim. Acta; citation_title=Cobalamin-dependent and cobalamin-independent methionine synthases: are there two solutions to the same chemical problem?; citation_author=RG Matthews; citation_volume=86; citation_publication_date=2003; citation_pages=3939-3954; citation_doi=10.1002/hlca.200390329; citation_id=CR52"/> <meta name="citation_reference" content="citation_journal_title=J. Am. Chem. Soc.; citation_title=Cobalt-carbon homolysis and bond dissociation energy studies of biological alkylcobalamins: methylcobalamin, including a >1015 Co-CH3 homolysis rate enhancement at 25 °C following one-electron reduction; citation_author=BD Martin, RG Finke; citation_volume=112; citation_publication_date=1990; citation_pages=2419-2420; citation_doi=10.1021/ja00162a053; citation_id=CR53"/> <meta name="citation_reference" content="citation_journal_title=J. Am. Chem. Soc.; citation_title=Methylcobalamin’s full- vs. half-strength cobalt-carbon σ bonds and bond dissociation enthalpies: A > 1015 Co-CH3 homolysis rate enhancement following one-antibonding-electron reduction of methlycobalamin; citation_author=BD Martin, RG Finke; citation_volume=114; citation_publication_date=1992; citation_pages=585-592; citation_doi=10.1021/ja00028a027; citation_id=CR54"/> <meta name="citation_reference" content="citation_journal_title=J. Am. Chem. Soc.; citation_title=Conversion of homocysteine to methionine by methionine synthase. A density functional study; citation_author=KP Jensen, U Ryde; citation_volume=125; citation_publication_date=2003; citation_pages=13970-13971; citation_doi=10.1021/ja034697a; citation_id=CR55"/> <meta name="citation_reference" content="citation_journal_title=Chem. Commun.; citation_title=Immobilised vitamin B12 as a biomimetic model for base-off/histidine-on coordination; citation_author=C Männel-Croisé, F Zelder; citation_volume=47; citation_publication_date=2011; citation_pages=11249-11251; citation_doi=10.1039/c1cc15093f; citation_id=CR56"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. Chem.; citation_title=Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl-CoA by Clostridium thermoaceticum; citation_author=SW Ragsdale, PA Lindahl, E Münck; citation_volume=262; citation_publication_date=1987; citation_pages=14289-14297; citation_id=CR57"/> <meta name="citation_reference" content="citation_journal_title=Science; citation_title=How a protein binds B12. A 3.0 A X-ray structure of B12-binding domains of methionine synthase; citation_author=C Drennan, S Huang, J Drummond, R Matthews, M Lidwig; citation_volume=266; citation_publication_date=1994; citation_pages=1669-1674; citation_doi=10.1126/science.7992050; citation_id=CR58"/> <meta name="citation_reference" content="citation_journal_title=ISME J.; citation_title=The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring Dehalococcoides mccartyi; citation_author=J Yan; citation_volume=10; citation_publication_date=2016; citation_pages=1092-1101; citation_doi=10.1038/ismej.2015.197; citation_id=CR59"/> <meta name="citation_reference" content="citation_journal_title=Annu. Rev. microbiology; citation_title=Total synthesis of acetate from CO2 by heterotrophic bacteria; citation_author=LG Ljungdahl, HG Wood; citation_volume=23; citation_publication_date=1969; citation_pages=515-538; citation_doi=10.1146/annurev.mi.23.100169.002503; citation_id=CR60"/> <meta name="citation_reference" content="citation_journal_title=Curr. Opin. Struct. Biol.; citation_title=Cobalamin-dependent and cobamide-dependent methyltransferases; citation_author=RG Matthews, M Koutmos, S Datta; citation_volume=18; citation_publication_date=2008; citation_pages=658-666; citation_doi=10.1016/j.sbi.2008.11.005; citation_id=CR61"/> <meta name="citation_reference" content="Hippler, B. & Thauer, R. K. The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH. FEBS Lett. 449, 165–168 (1999)."/> <meta name="citation_reference" content="citation_journal_title=Curr. Opin. Biotechnol.; citation_title=Fundamentals of methanogenic pathways that are key to the biomethanation of complex biomass; citation_author=JG Ferry; citation_volume=22; citation_publication_date=2011; citation_pages=351-357; citation_doi=10.1016/j.copbio.2011.04.011; citation_id=CR63"/> <meta name="citation_reference" content="citation_journal_title=Eur. J. Biochem.; citation_title=Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum; citation_author=S Länge, G Fuchs; citation_volume=163; citation_publication_date=1987; citation_pages=147-154; citation_doi=10.1111/j.1432-1033.1987.tb10748.x; citation_id=CR64"/> <meta name="citation_reference" content="citation_journal_title=BMC Bioinfomatics; citation_title=I-TASSER server for protein 3D structure prediction; citation_author=Y Zhang; citation_volume=9; citation_publication_date=2008; citation_pages=1-8; citation_doi=10.1186/1471-2105-9-1; citation_id=CR65"/> <meta name="citation_reference" content="DeLano, W. L. PyMol: an open-source molecular graphics tool. CCP4 Newsletter on Protein Crystallography. 40, 82–92 (2002)."/> <meta name="citation_reference" content="citation_journal_title=Mol. Biol. Evol.; citation_title=MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets; citation_author=S Kumar, G Stecher, K Tamura; citation_volume=33; citation_publication_date=2016; citation_pages=1870-1874; citation_doi=10.1093/molbev/msw054; citation_id=CR67"/> <meta name="citation_reference" content="citation_journal_title=Nucleic Acids Res.; citation_title=MUSCLE: multiple sequence alignment with high accuracy and high throughput; citation_author=RC Edgar; citation_volume=32; citation_publication_date=2004; citation_pages=1792-1797; citation_doi=10.1093/nar/gkh340; citation_id=CR68"/> <meta name="citation_reference" content="citation_journal_title=Comput. Appl. Biosci.; citation_title=The rapid generation of mutation data matrices from protein sequences; citation_author=DT Jones, WR Taylor, JM Thornton; citation_volume=8; citation_publication_date=1992; citation_pages=275-282; citation_id=CR69"/> <meta name="citation_reference" content="Zuckerkandl, E. & Pauling, L. In Evolving genes and proteins (Elsevier), pp. 97–166 (1965)."/> <meta name="citation_reference" content="citation_journal_title=Ann. NY. Acad. Sci.; citation_title=Disc electrophoresis-I: background and theory; citation_author=L Ornstein, BJ Davis; citation_volume=121; citation_publication_date=1964; citation_pages=321-349; citation_doi=10.1111/j.1749-6632.1964.tb14207.x; citation_id=CR71"/> <meta name="citation_reference" content="citation_journal_title=Curr. Protoc. Bioinforma.; citation_title=Employing ProteoWizard to convert raw mass spectrometry data; citation_author=JD Holman, DL Tabb, P Mallick; citation_volume=46; citation_publication_date=2014; citation_pages=13.24.1-9; citation_doi=10.1002/0471250953.bi1324s46; citation_id=CR72"/> <meta name="citation_reference" content="citation_journal_title=Proteom.; citation_title=SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches; citation_author=M Vaudel, H Barsnes, FS Berven, A Sickmann, L Martens; citation_volume=11; citation_publication_date=2011; citation_pages=996-999; citation_doi=10.1002/pmic.201000595; citation_id=CR73"/> <meta name="citation_reference" content="citation_journal_title=Nat. Biotechnol.; citation_title=PeptideShaker enables reanalysis of MS-derived proteomics data sets; citation_author=M Vaudel; citation_volume=33; citation_publication_date=2015; citation_pages=22-24; citation_doi=10.1038/nbt.3109; citation_id=CR74"/> <meta name="citation_reference" content="citation_journal_title=Eur. J. Biochem.; citation_title=Methanol:coenzyme M methyltransferase from Methanosarcina barkeri - substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin; citation_author=K Sauer, RK Thauer; citation_volume=261; citation_publication_date=1999; citation_pages=674-681; citation_doi=10.1046/j.1432-1327.1999.00355.x; citation_id=CR75"/> <meta name="citation_reference" content="citation_journal_title=J. Biol. Chem.; citation_title=Purification and properties of pancreatic glycine N-methyltransferase; citation_author=EJ Yeo, C Wagner; citation_volume=267; citation_publication_date=1992; citation_pages=24669-24674; citation_id=CR76"/> <meta name="citation_reference" content="citation_journal_title=J. Agric. Food Chem.; citation_title=Development of a simplified method for the determination of folates in baker’s yeast by HPLC with ultraviolet and fluorescence detection; citation_author=JDM Patring, JA Jastrebova, SB Hjortmo, TA Andlid, IM Jägerstad; citation_volume=53; citation_publication_date=2005; citation_pages=2406-2411; citation_doi=10.1021/jf048083g; citation_id=CR77"/> <meta name="citation_reference" content="citation_journal_title=Mol. Sys Biol.; citation_title=Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection; citation_author=T Baba; citation_volume=2; citation_publication_date=2006; citation_pages=1-11; citation_id=CR78"/> <meta name="citation_reference" content="Harwood, C. R. & Cutting, S. M. In Molecular biological methods in Bacillus, edited by C. R. Harwood & S. M. Cutting (Wiley, Chichester), Vol. 1, p. 548 (1990)."/> <meta name="citation_reference" content="citation_journal_title=Eur. J. Biochem.; citation_title=Vitamin-B12-Independent Methionine Synthase from a Higher Plant (Catharanthus Roseus); citation_author=J Eichel, JC González, M Hotze, RG Matthews, J Schröder; citation_volume=230; citation_publication_date=1995; citation_pages=1053-1058; citation_doi=10.1111/j.1432-1033.1995.tb20655.x; citation_id=CR80"/> <meta name="citation_author" content="Deobald, Darja"/> <meta name="citation_author_institution" content="Leipzig University, Institute of Biochemistry, Brüderstraße 34, Leipzig, Germany"/> <meta name="citation_author_institution" content="Helmholtz Centre for Environmental Research – UFZ, Isotope Biogeochemistry, Permoserstraße 15, Leipzig, Germany"/> <meta name="citation_author" content="Hanna, Rafael"/> <meta name="citation_author_institution" content="Leipzig University, Institute of Biochemistry, Brüderstraße 34, Leipzig, Germany"/> <meta name="citation_author_institution" content="Freiburg University, Institute of Pharmaceutical Sciences, Stefan-Meier-Straße 19, Freiburg im Breisgau, Germany"/> <meta name="citation_author" content="Shahryari, Shahab"/> <meta name="citation_author_institution" content="Helmholtz Centre for Environmental Research – UFZ, Isotope Biogeochemistry, Permoserstraße 15, Leipzig, Germany"/> <meta name="citation_author" content="Layer, Gunhild"/> <meta name="citation_author_institution" content="Leipzig University, Institute of Biochemistry, Brüderstraße 34, Leipzig, Germany"/> <meta name="citation_author_institution" content="Freiburg University, Institute of Pharmaceutical Sciences, Stefan-Meier-Straße 19, Freiburg im Breisgau, Germany"/> <meta name="citation_author" content="Adrian, Lorenz"/> <meta name="citation_author_institution" content="Helmholtz Centre for Environmental Research – UFZ, Isotope Biogeochemistry, Permoserstraße 15, Leipzig, Germany"/> <meta name="citation_author_institution" content="Technische Universität Berlin, Chair of Geobiotechnology, Ackerstraße 76, Berlin, Germany"/> <meta name="access_endpoint" content="https://www.nature.com/platform/readcube-access"/> <meta name="twitter:site" content="@SciReports"/> <meta name="twitter:card" content="summary_large_image"/> <meta name="twitter:image:alt" content="Content cover image"/> <meta name="twitter:title" content="Identification and characterization of a bacterial core methionine synthase"/> <meta name="twitter:description" content="Scientific Reports - Identification and characterization of a bacterial core methionine synthase"/> <meta name="twitter:image" content="https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig1_HTML.png"/> <meta property="og:url" content="https://www.nature.com/articles/s41598-020-58873-z"/> <meta property="og:type" content="article"/> <meta property="og:site_name" content="Nature"/> <meta property="og:title" content="Identification and characterization of a bacterial core methionine synthase - Scientific Reports"/> <meta property="og:image" content="https://media.springernature.com/m685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig1_HTML.png"/> <script> window.eligibleForRa21 = 'false'; </script> </head> <body class="article-page"> <noscript><iframe src="https://www.googletagmanager.com/ns.html?id=GTM-MRVXSHQ" height="0" width="0" style="display:none;visibility:hidden"></iframe></noscript> <div class="position-relative cleared z-index-50 background-white" data-test="top-containers"> <a class="c-skip-link" href="#content">Skip to main content</a> <div class="c-grade-c-banner u-hide"> <div class="c-grade-c-banner__container"> <p>Thank you for visiting nature.com. 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characterization of a bacterial core methionine synthase</h1> <ul class="c-article-author-list c-article-author-list--short" data-test="authors-list" data-component-authors-activator="authors-list"><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Darja-Deobald-Aff1-Aff2" data-author-popup="auth-Darja-Deobald-Aff1-Aff2" data-author-search="Deobald, Darja">Darja Deobald</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a></sup>, </li><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Rafael-Hanna-Aff1-Aff3" data-author-popup="auth-Rafael-Hanna-Aff1-Aff3" data-author-search="Hanna, Rafael">Rafael Hanna</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff3">3</a></sup>, </li><li class="c-article-author-list__item 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data-track-label="link"><i data-test="journal-title">Scientific Reports</i></a> <b data-test="journal-volume"><span class="u-visually-hidden">volume</span> 10</b>, Article number: <span data-test="article-number">2100</span> (<span data-test="article-publication-year">2020</span>) <a href="#citeas" class="c-article-info-details__cite-as u-hide-print" data-track="click" data-track-action="cite this article" data-track-label="link">Cite this article</a> </p> <div class="c-article-metrics-bar__wrapper u-clear-both"> <ul class="c-article-metrics-bar u-list-reset"> <li class=" c-article-metrics-bar__item" data-test="access-count"> <p class="c-article-metrics-bar__count">7162 <span class="c-article-metrics-bar__label">Accesses</span></p> </li> <li class="c-article-metrics-bar__item" data-test="altmetric-score"> <p class="c-article-metrics-bar__count">3 <span class="c-article-metrics-bar__label">Altmetric</span></p> </li> <li class="c-article-metrics-bar__item"> <p 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href="/subjects/transferases" data-track="click" data-track-action="view subject" data-track-label="link">Transferases</a></li> </ul> </div> </div> <div class="c-article-body"> <section aria-labelledby="Abs1" data-title="Abstract" lang="en"><div class="c-article-section" id="Abs1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Abs1">Abstract</h2><div class="c-article-section__content" id="Abs1-content"><p>Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In <i>Dehalococcoides mccartyi</i> strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class <i>Dehalococcoidia</i> lack <i>metF</i> encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-<i>metE</i> gene. We heterologously expressed core-MetE<sub>CBDB</sub> in <i>E. coli</i> and purified the 38 kDa protein. Core-MetE<sub>CBDB</sub> exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (<i>K</i><sub>M</sub> ≈ 240 µM) and L-homocysteine (<i>K</i><sub>M</sub> ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a <i>k</i><sub>cat</sub> ≈ 60 s<sup>−1</sup>. Core-MetE<sub>CBDB</sub> did not functionally complement <i>metE</i>-deficient <i>E. coli</i> strain DH5α (<i>ΔmetE::kan</i>) suggesting that core-MetE<sub>CBDB</sub> and the canonical MetE enzyme from <i>E. coli</i> have different enzymatic specificities also <i>in vivo</i>. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain.</p></div></div></section> <section aria-labelledby="inline-recommendations" data-title="Inline Recommendations" class="c-article-recommendations" data-track-component="inline-recommendations"> <h3 class="c-article-recommendations-title" id="inline-recommendations">Similar content being viewed by others</h3> <div class="c-article-recommendations-list"> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41559-022-01857-1/MediaObjects/41559_2022_1857_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41559-022-01857-1?fromPaywallRec=false" data-track="select_recommendations_1" data-track-context="inline recommendations" data-track-action="click recommendations inline - 1" data-track-label="10.1038/s41559-022-01857-1">An early origin of iron–sulfur cluster biosynthesis machineries before Earth oxygenation </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__date">15 September 2022</span> </div> </div> </article> </div> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41598-024-65342-4/MediaObjects/41598_2024_65342_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41598-024-65342-4?fromPaywallRec=false" data-track="select_recommendations_2" data-track-context="inline recommendations" data-track-action="click recommendations inline - 2" data-track-label="10.1038/s41598-024-65342-4">Insights into the genomic and functional divergence of <i>NAT</i> gene family to serve microbial secondary metabolism </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__access-type">Open access</span> <span class="c-article-meta-recommendations__date">28 June 2024</span> </div> </div> </article> </div> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41396-023-01477-y/MediaObjects/41396_2023_1477_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41396-023-01477-y?fromPaywallRec=false" data-track="select_recommendations_3" data-track-context="inline recommendations" data-track-action="click recommendations inline - 3" data-track-label="10.1038/s41396-023-01477-y">Stepwise pathway for early evolutionary assembly of dissimilatory sulfite and sulfate reduction </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__access-type">Open access</span> <span class="c-article-meta-recommendations__date">19 July 2023</span> </div> </div> </article> </div> </div> </section> <script> window.dataLayer = window.dataLayer || []; window.dataLayer.push({ recommendations: { recommender: 'semantic', model: 'specter', policy_id: 'NA', timestamp: 1739790099, embedded_user: 'null' } }); </script> <div class="main-content"> <section data-title="Introduction"><div class="c-article-section" id="Sec1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec1">Introduction</h2><div class="c-article-section__content" id="Sec1-content"><p>Methionine plays an essential role as proteinogenic amino acid in all domains of life, as an initiation amino acid in protein translation<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208 (1999)." href="/articles/s41598-020-58873-z#ref-CR1" id="ref-link-section-d466690512e451">1</a></sup> and as a precursor in the formation of cysteine, carnitine, taurine and lecithin<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 2" title="Stipanuk, M. H. Metabolism of sulfur-containing amino acids. Ann. Rev. Nutr. 6, 179–209 (1986)." href="/articles/s41598-020-58873-z#ref-CR2" id="ref-link-section-d466690512e455">2</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 3" title="Stipanuk, M. H. & Ueki, I. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur. J. Inherit. Metab. Dis. 34, 17–32 (2011)." href="/articles/s41598-020-58873-z#ref-CR3" id="ref-link-section-d466690512e458">3</a></sup>. Moreover, methionine can be converted to <i>S-</i>adenosyl-L-methionine (SAM)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 4" title="Cantoni, G. L. S-adenosylmethionine; a new intermediate formed enzymatically from L-methionine and adenosinetriphosphate. J. Biol. Chem. 204, 403–416 (1953)." href="/articles/s41598-020-58873-z#ref-CR4" id="ref-link-section-d466690512e465">4</a></sup>, which represents an activated methyl group donor for many fundamental cellular processes<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Gophna, U., Bapteste, E., Doolittle, W. F., Biran, D. & Ron, E. Z. Evolutionary plasticity of methionine biosynthesis. Gene 355, 48–57 (2005)." href="/articles/s41598-020-58873-z#ref-CR5" id="ref-link-section-d466690512e469">5</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Frey, P. A., Ballinger, M. D. & Reed, G. H. S-adenosylmethionine. A ‘poor man’s coenzyme B12’ in the reaction of lysine 2,3-aminomutase. Biochm Soc. Trans. 26, 304–310 (1998)." href="/articles/s41598-020-58873-z#ref-CR6" id="ref-link-section-d466690512e472">6</a></sup>. The final step in methionine <i>de novo</i> synthesis, the methylation of homocysteine to methionine, is catalyzed by different types of methionine synthases including cobalamin-dependent (MetH) and cobalamin-independent methionine synthase (MetE). Some bacteria, <i>e.g. Escherichia coli</i>, possess genes for both enzymes<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 7" title="Foster, M. A., Tejerina, G., Guest, J. R. & Woods, D. D. Two enzymic mechanisms for the methylation of homocysteine by extracts of Escherichia coli. Biochem. 92, 476–488 (1964)." href="/articles/s41598-020-58873-z#ref-CR7" id="ref-link-section-d466690512e483">7</a></sup> and repress the expression of <i>metE</i> in the presence of vitamin B<sub>12</sub><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 8" title="Weissbach, H. & Brot, N. Regulation of methionine synthesis in Escherichia coli. Mol. Microbiol. 5, 1593–1597 (1991)." href="/articles/s41598-020-58873-z#ref-CR8" id="ref-link-section-d466690512e491">8</a></sup>. Homocysteine methylation in mammals is catalyzed by mammalian methionine synthases (mMS) similar to bacterial MetH<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 9" title="Wolthers, K. R. et al. Crystal structure and solution characterization of the activation domain of human methionine synthase. FEBS J. 274, 738–750 (2007)." href="/articles/s41598-020-58873-z#ref-CR9" id="ref-link-section-d466690512e496">9</a></sup>, betaine-L-homocysteine-<i>S</i>-methyltransferase (BHMT) or <i>S-</i>methyl-L-methionine-L-homocysteine-<i>S</i>-methyltransferase (also known as BHMT-2)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Szegedi, S. S., Castro, C. C., Koutmos, M. & Garrow, T. A. Betaine-homocysteine-S-methyltransferase-2 is an S-methylmethionine-homocysteine methyltransferase. J. Biol. Chem. 283, 8939–8945 (2008)." href="/articles/s41598-020-58873-z#ref-CR10" id="ref-link-section-d466690512e509">10</a></sup>. Fungi and plants encode exclusively MetE<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Kacprzak, M. M., Lewandowska, I., Matthews, R. G. & Paszewski, A. Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans. Biochem. J. 376, 517–524 (2003)." href="/articles/s41598-020-58873-z#ref-CR11" id="ref-link-section-d466690512e513">11</a></sup> or BHMT-2<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 12" title="Bourgis, F. et al. S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase. Plant. Cell 11, 1485–1497 (1999)." href="/articles/s41598-020-58873-z#ref-CR12" id="ref-link-section-d466690512e518">12</a></sup>. All known methionine synthase types contain a zinc ion in the active site that is essential for homocysteine binding and methyl group transfer<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1, 332–339 (1997)." href="/articles/s41598-020-58873-z#ref-CR13" id="ref-link-section-d466690512e522">13</a></sup>.</p><p>MetH (EC 2.1.1.13) catalyzes the methyl transfer from 5-methyl-tetrahydrofolate-monoglutamate (5-methyl-THF-Glu) to homocysteine. MetH from <i>E. coli</i> is a large monomeric protein of 1,227 amino acids (136 kDa) and is composed of four functional domains<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 14" title="Datta, S., Koutmos, M., Pattridge, K. A., Ludwig, M. L. & Matthews, R. G. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor. Proc. Natl Acad. Sci. USA 105, 4115–4120 (2008)." href="/articles/s41598-020-58873-z#ref-CR14" id="ref-link-section-d466690512e532">14</a></sup>. In the catalytic cycle the methyl group of methylcob(III)alamin is transferred to homocysteine forming cob(I)alamin and methionine. Subsequently, cob(I)alamin is remethylated using 5-methyl-THF-Glu as the methyl group donor regenerating methylcob(III)alamin. For reactivation of cob(II)alamin to methylcob(III)alamin, which is generated in a side-reaction approximately once in 2,000 turnovers<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Drummond, J. T., Huang, S., Blumenthal, R. M. & Matthews, R. G. Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli. Biochem. 32, 9290–9295 (1993)." href="/articles/s41598-020-58873-z#ref-CR15" id="ref-link-section-d466690512e536">15</a></sup>, SAM is required<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 16" title="Goulding, C. W., Postigo, D. & Matthews, R. G. Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine. Biochem. 36, 8082–8091 (1997)." href="/articles/s41598-020-58873-z#ref-CR16" id="ref-link-section-d466690512e540">16</a></sup>.</p><p>Canonical MetE proteins (EC 2.1.1.14) are described as a family of zinc-containing metalloenzymes sharing no sequence similarity with MetH<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208 (1999)." href="/articles/s41598-020-58873-z#ref-CR1" id="ref-link-section-d466690512e547">1</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1, 332–339 (1997)." href="/articles/s41598-020-58873-z#ref-CR13" id="ref-link-section-d466690512e550">13</a></sup>. They catalyze the methylation of homocysteine using 5-methyl-THF-Glu<sub>n</sub> (n ≥ 3) as methyl donor without the involvement of cobalamin. MetE in <i>E. coli</i> is a protein of 753 amino acid residues (85 kDa) that is composed of two homologous parts connected by a linker region (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1b</a>), suggesting that the domains have evolved by gene duplication of a sequence encoding a smaller protein of approximately 340 amino acid residues<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Ferrer, J.-L., Ravanel, S., Robert, M. & Dumas, R. Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate. J. Biol. Chem. 279, 44235–44238 (2004)." href="#ref-CR17" id="ref-link-section-d466690512e562">17</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Fu, T.-M. et al. Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans. A dynamic zinc-inversion model. J. Mol. Biol. 412, 688–697 (2011)." href="#ref-CR18" id="ref-link-section-d466690512e562_1">18</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 19" title="Pejchal, R. & Ludwig, M. L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. PLoS Biol. 3, 254–265 (2005)." href="/articles/s41598-020-58873-z#ref-CR19" id="ref-link-section-d466690512e565">19</a></sup>. We here refer to the canonical <i>E. coli</i>-type MetE as ‘tandem-repeat MetE’ (tr-MetE). The active site of tr-MetE is located within the C-terminal part, where the zinc ion is coordinated by one histidine, two cysteine and one glutamate residue. The binding site for methyl-THF is in the cleft between the two domains of tr-MetE<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 19" title="Pejchal, R. & Ludwig, M. L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. PLoS Biol. 3, 254–265 (2005)." href="/articles/s41598-020-58873-z#ref-CR19" id="ref-link-section-d466690512e573">19</a></sup>.</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-1" data-title="Figure 1"><figure><figcaption><b id="Fig1" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 1</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/1" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig1_HTML.png?as=webp"><img aria-describedby="Fig1" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig1_HTML.png" alt="figure 1" loading="lazy" width="685" height="646"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-1-desc"><p>Bioinformatic analysis of the core-MetE<sub>CBDB</sub> from <i>Dehalococcoides mccartyi</i> strain CBDB1. <b>(a)</b> Maximum-Likelihood phylogenetic tree of MetE representatives was generated with MEGA7<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 67" title="Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016)." href="/articles/s41598-020-58873-z#ref-CR67" id="ref-link-section-d466690512e595">67</a></sup>. Multiple amino acid sequence alignments of full length with deletion of gaps (MUSCLE algorithm) were used to generate the tree. The analysis involved 39 amino acid sequences including the C-terminus of tandem-repeat methionine synthases (tr-MetE) from bacteria (brown colors) and yeast (green) as well as core-MetEs from archaea (blue colors), <i>Chloroflexi</i> (red) and <i>Clostridiales</i> (brown). The gene loci are in brackets. <b>(b)</b> The crystal structure of core-MetE<sub>CBDB</sub> was calculated with the I-TASSER server<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 65" title="Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinfomatics 9, 1–8 (2008)." href="/articles/s41598-020-58873-z#ref-CR65" id="ref-link-section-d466690512e611">65</a></sup>. Overlay of the crystal structure of tr-MetE from <i>Neurospora crassa</i> (PDB No. 4ZTX, grey) with the structural model obtained for core-MetE<sub>CBDB</sub> from <i>D. mccartyi</i> strain CBDB1 (green) was obtained with PyMOL<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 66" title="DeLano, W. L. PyMol: an open-source molecular graphics tool. CCP4 Newsletter on Protein Crystallography. 40, 82–92 (2002)." href="/articles/s41598-020-58873-z#ref-CR66" id="ref-link-section-d466690512e624">66</a></sup>. Core-MetE<sub>CBDB</sub> matches the C-terminal part of tr-MetE<sub>Ncra</sub> (C-score = −0.27) but lacks the N-terminal part and the linker region. <b>(c)</b> Amino acid sequence alignment of selected MetE proteins. The Zn<sup>2+</sup>-binding site HXCX<sub>n</sub>C (red) is conserved in annotated tr-MetEs and also in core-MetE homologs. Core-MetE<sub>CBDB</sub>: core-MetE from <i>D. mccartyi</i> strain CBDB1, coreMetE<sub>Dehly</sub>: core-MetE from <i>Dehalogenimonas lykanthroporepellens</i>, core-MetE<sub>MMKA</sub>: core-MetE from <i>Methanococcus maripaludis</i>, tr-MetE<sub>ECDH</sub>: tandem-repeat MetE from <i>Escherichia coli</i> DH10B, tr-MetE<sub>Sau</sub>: tandem-repeat MetE from <i>Staphylococcus aureus</i>.</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/1" data-track-dest="link:Figure1 Full size image" aria-label="Full size image figure 1" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p><i>Dehalococcoides mccartyi</i> strain CBDB1 is an obligately anaerobic, mesophilic bacterium belonging to the phylum <i>Chloroflexi</i>, class <i>Dehalococcoidia</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 20" title="Adrian, L., Szewzyk, U., Wecke, J. & Görisch, H. Bacterial dehalorespiration with chlorinated benzenes. Nat. 408, 580–583 (2000)." href="/articles/s41598-020-58873-z#ref-CR20" id="ref-link-section-d466690512e684">20</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 21" title="Hölscher, T., Gorisch, H. & Adrian, L. Reductive dehalogenation of chlorobenzene congeners in cell extracts of Dehalococcoides sp. strain CBDB1. Appl. Env. Microbiol. 69, 2999–3001 (2003)." href="/articles/s41598-020-58873-z#ref-CR21" id="ref-link-section-d466690512e687">21</a></sup>. <i>Dehalococcoides</i> species are well known for their ability to use a wide range of persistent and toxic halogenated organic compounds as terminal electron acceptor in anaerobic respiration (“organohalide respiration”) with hydrogen as electron donor<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 22" title="Löffler, F. E. et al. Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi. Int. J. Syst. Evol. Microbiol. 63, 625–635 (2013)." href="/articles/s41598-020-58873-z#ref-CR22" id="ref-link-section-d466690512e694">22</a></sup>. Strain CBDB1 encodes one of the largest numbers of B<sub>12</sub>-dependent proteins in known prokaryotes<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 23" title="Zhang, Y., Rodionov, D. A., Gelfand, M. S. & Gladyshev, V. N. Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC genomics 10, 78 (2009)." href="/articles/s41598-020-58873-z#ref-CR23" id="ref-link-section-d466690512e701">23</a></sup>. The most prominent representatives of B<sub>12</sub>-dependent proteins in strain CBDB1 are reductive dehalogenases that are responsible for the reduction of halogenated pollutants as a terminal electron acceptor<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 24" title="Adrian, L., Rahnenführer, J., Gobom, J. & Hölscher, T. Identification of a chlorobenzene reductive dehalogenase in Dehalococcoides sp. strain CBDB1. Appl. Env. Microbiol. 73, 7717–7724 (2007)." href="/articles/s41598-020-58873-z#ref-CR24" id="ref-link-section-d466690512e707">24</a></sup>. Vitamin B<sub>12</sub> in the medium is essential for the growth of <i>Dehalococcoides</i> strains<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 25" title="Schipp, C. J., Marco-Urrea, E., Kublik, A., Seifert, J. & Adrian, L. Organic cofactors in the metabolism of Dehalococcoides mccartyi strains. Philos. Trans. R. Soc. Lond. 368, 20120321 (2013)." href="/articles/s41598-020-58873-z#ref-CR25" id="ref-link-section-d466690512e717">25</a></sup> because <i>Dehalococcoides</i> do not contain many of the genes for <i>de novo</i> biosynthesis of cobalamin<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Kube, M. et al. Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1. Nat. Biotechnol. 23, 1269–1273 (2005)." href="/articles/s41598-020-58873-z#ref-CR26" id="ref-link-section-d466690512e727">26</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 27" title="Seshadri, R. et al. Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes. Sci. 307, 105–108 (2005)." href="/articles/s41598-020-58873-z#ref-CR27" id="ref-link-section-d466690512e730">27</a></sup>. However, <i>Dehalococcoides</i> strains encode corrinoid-specific ABC-transporter and enzymes for the late B<sub>12</sub> biosynthesis pathway enabling them to incorporate corrinoid precursors from the environment and to modify them to cobalamin<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 28" title="Yi, S. et al. Versatility in corrinoid salvaging and remodeling pathways supports corrinoid-dependent metabolism in Dehalococcoides mccartyi. Appl. Env. Microbiol. 78, 7745–7752 (2012)." href="/articles/s41598-020-58873-z#ref-CR28" id="ref-link-section-d466690512e740">28</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 29" title="Yan, J., Ritalahti, K. M., Wagner, D. D. & Löffler, F. E. Unexpected specificity of interspecies cobamide transfer from Geobacter spp. to organohalide-respiring Dehalococcoides mccartyi strains. Appl. Env. Microbiol. 78, 6630–6636 (2012)." href="/articles/s41598-020-58873-z#ref-CR29" id="ref-link-section-d466690512e743">29</a></sup>. Although none of the known <i>D. mccartyi</i> genomes contains full gene homologs of <i>metE</i>, <i>metH, bhmt</i> or <i>bhmt-2</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Kube, M. et al. Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1. Nat. Biotechnol. 23, 1269–1273 (2005)." href="/articles/s41598-020-58873-z#ref-CR26" id="ref-link-section-d466690512e758">26</a></sup>, <i>D. mccartyi</i> strains synthesize methionine <i>de novo</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 24" title="Adrian, L., Rahnenführer, J., Gobom, J. & Hölscher, T. Identification of a chlorobenzene reductive dehalogenase in Dehalococcoides sp. strain CBDB1. Appl. Env. Microbiol. 73, 7717–7724 (2007)." href="/articles/s41598-020-58873-z#ref-CR24" id="ref-link-section-d466690512e768">24</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 30" title="Tang, Y. J. et al. Investigation of carbon metabolism in “Dehalococcoides ethenogenes” strain 195 by use of isotopomer and transcriptomic analyses. J. Bacteriol. 191, 5224–5231 (2009)." href="/articles/s41598-020-58873-z#ref-CR30" id="ref-link-section-d466690512e771">30</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 31" title="Marco-Urrea, E., Seifert, J., Bergen, Mvon & Adrian, L. Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1. J. Bacteriol. 194, 4169–4177 (2012)." href="/articles/s41598-020-58873-z#ref-CR31" id="ref-link-section-d466690512e774">31</a></sup>. Zhuang <i>et al</i>. demonstrated that acetyl-CoA donates the C<sub>2</sub>-methyl group for an unconventional methionine biosynthesis pathway independent from methylene-tetrahydrofolate reductase (MTHFR). All <i>D. mccartyi</i> species sequenced so far, lack <i>metF</i> encoding for MTHFR that reduces 5,10-methylene-THF-Glu to 5-methyl-THF-Glu. Furthermore, <i>D. mccartyi</i> strain 195 was not able to incorporate 5-methyl-THF from the environment<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 32" title="Zhuang, W.-Q. et al. Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi. Proc. Natl Acad. Sci. USA 111, 6419–6424 (2014)." href="/articles/s41598-020-58873-z#ref-CR32" id="ref-link-section-d466690512e793">32</a></sup>.</p><p>Here, we found that locus cbdbA481 (NCBI accession number CAI82680) of <i>D. mccartyi</i> strain CBDB1 encodes a 343 amino acid protein that is homologous to the C-terminus of canonical tr-MetE and similar to methylcobalamin:homocysteine methyltransferase (core-MetE<sub>MTH</sub>) of the methanogenic archaeum <i>Methanobacterium thermoautotrophicum</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e808">33</a></sup>. In our study we provide biochemical and genetic evidence that the locus cbdbA481 encodes a novel type of bacterial methionine synthase that appears to be an anciently derived MetE-related methionine synthase obtaining its methyl group from an external corrinoid rather than from folate. Together with archaeal methylcobalamin:homocysteine methyltransferases and bacterial homologs the gene product of locus cbdbA481 forms a new group of basal methionine synthases, referred to as core-MetE in the following.</p></div></div></section><section data-title="Results"><div class="c-article-section" id="Sec2-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec2">Results</h2><div class="c-article-section__content" id="Sec2-content"><h3 class="c-article__sub-heading" id="Sec3">Bioinformatic analysis of locus cbdbA481 in the genome of <i>D. mccartyi</i> strain CBDB1</h3><p><i>D. mccartyi</i> strains are able to synthesize methionine <i>de novo</i>, although <i>D. mccartyi</i> genomes do not contain gene homologs of <i>metE, metH, bhmt</i> or <i>bhmt</i>-2<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Kube, M. et al. Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1. Nat. Biotechnol. 23, 1269–1273 (2005)." href="/articles/s41598-020-58873-z#ref-CR26" id="ref-link-section-d466690512e842">26</a></sup>. In the KEGG (Kyoto Encyclopedia of Genes and Genomes) database several enzymes of the <i>Dehalococcoides</i> methionine metabolism are annotated<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000)." href="#ref-CR34" id="ref-link-section-d466690512e850">34</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. & Tanabe, M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 44, 457–462 (2016)." href="#ref-CR35" id="ref-link-section-d466690512e850_1">35</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 36" title="Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45, 353–361 (2017)." href="/articles/s41598-020-58873-z#ref-CR36" id="ref-link-section-d466690512e853">36</a></sup>. The loci cbdbA476 and cbdbA477 in the genome of strain CBDB1 are annotated as SAM synthetase (EC 2.5.1.6) and <i>S</i>-adenosyl-L-homocysteinase (EC 3.3.1.1), respectively. Since methionine biosynthesis and SAM metabolism are biochemically closely linked, we started our search for a methionine synthase gene in the genome of strain CBDB1 by inspecting the direct neighborhood of the loci cbdbA476 and cbdbA477. Locus cbdbA481 located in the same operon encodes a 343 amino acid polypeptide (here referred to as core-MetE<sub>CBDB</sub>) with a calculated molecular mass of 38 kDa that has sequence similarity with the C-terminal half of canonical tandem-repeat MetE (tr-MetE) proteins. Accordingly, the calculated mass of core-MetE<sub>CBDB</sub> is only about half the size of tr-MetE proteins (<i>e.g</i>. tr-MetE<sub>Eco</sub> of <i>E. coli</i>). Phylogenetic analysis of core-MetE<sub>CBDB</sub> together with 38 other protein sequences including annotated tr-MetE representatives, archaeal methylcobalamin:homocysteine methyltransferase homologs (core-MetE<sub>Archaea</sub>) and <i>Chloroflexi</i> sequences with high sequence similarity to core-MetE<sub>CBDB</sub> indicates that core-MetE<sub>CBDB</sub> is the prototype of a new bacterial cluster of short MetE sequences (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>). This new cluster is phylogenetically well separated from the C-termini of tr-MetE proteins and core-MetE<sub>Archaea</sub> (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>, blue colors)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e893">33</a></sup>. Core-MetE proteins are widely distributed in microorganisms with strongly conserved ancient traits (archaea, <i>Clostridiales, Dehalococcoidia</i> and <i>Chloroflexia</i> classes). Within the archaea, only <i>Haloquadratum</i> spp. encode the tr-MetE, whereas many archaea encode a core-MetE homolog (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>, blue colors). Bacterial and archaeal core-MetE form a paraphyletic group (excluding the tr-MetE sequences), probably evolved before LUCA (last universal common ancestor) and branched into two groups. Tr-MetEs appear to have evolved from archaeal core-MetE (<i>e.g</i>. core-MetE in <i>Clostridium kluyveri</i> and <i>C. oryzae</i>) (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>).</p><p>The computational structural model of core-MetE<sub>CBDB</sub> (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1b</a>, green) resembles the C-terminal domain of annotated tr-MetE proteins, bearing the highest similarity to methionine synthase from <i>Neurospora crassa</i> (C-score = −0.27, identity = 21% and RMSD = 3.52) (PDB No. 4ZTX)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 37" title="Wheatley, R. W., Ng, K. K. S. & Kapoor, M. Fungal cobalamin-independent methionine synthase: Insights from the model organism, Neurospora crassa. Arch. Biochem. Biophys. 590, 125–137 (2016)." href="/articles/s41598-020-58873-z#ref-CR37" id="ref-link-section-d466690512e934">37</a></sup>. Compared to tr-MetE proteins, core-MetE<sub>CBDB</sub> lacks the N-terminal domain described to be responsible for 5-methyl-THF binding and the linker region between the C- and N-terminal parts (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1b</a>, grey). An amino acid alignment of core-MetE<sub>CBDB</sub> with core-MetE<sub>Dehly</sub> from <i>Dehalogenimonas lykanthroporepellens</i>, core-MetE<sub>MMKA</sub> from <i>Methanococcus maripaludis</i> KA1 and the C-terminal halves of the tr-MetE<sub>ECDH</sub> from <i>E. coli</i> DH10B and tr-MetE<sub>Sau</sub> from <i>Staphylococcus aureus</i> shows that the zinc binding motif HXCX<sub>n</sub>C, essential for L-homocysteine binding and activation<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1, 332–339 (1997)." href="/articles/s41598-020-58873-z#ref-CR13" id="ref-link-section-d466690512e969">13</a></sup>, is conserved in core-MetE<sub>CBDB</sub> (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1c</a>). The computational model indicates that in strain CBDB1, zinc is coordinated in a tetrahedral fashion by His215, Cys217 and Cys312 (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1c</a>) which are conserved among all MetEs and by Asp236. In contrast, zinc of tr-MetE from <i>N. crassa</i> is bound by histidine, two cysteine and one glutamate residue.</p><h3 class="c-article__sub-heading" id="Sec4">Heterologous production and purification of core-MetE<sub>CBDB</sub> </h3><p>To study the function of core-MetE<sub>CBDB</sub> in detail, the recombinant protein was heterologously produced in <i>E. coli</i> and purified. First, production and purification attempts were conducted for a C-terminally Streptavidin-tagged core-MetE<sub>CBDB</sub> using affinity chromatography for purification. However, native polyacrylamide gel electrophoresis (PAGE) indicated misfolding of the protein (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">1a,b</a>). Therefore, core-MetE<sub>CBDB</sub> was produced without a tag in <i>E. coli</i> and purified using anion exchange chromatography. The purified protein exhibited a molecular mass of approximately 38 kDa, as determined by SDS-PAGE (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">1c</a>). The identity of core-MetE<sub>CBDB</sub> was verified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (47 validated unique peptides, 87% coverage). Purified core-MetE<sub>CBDB</sub> was in its monomeric form as indicated by native PAGE (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">1d</a>) and analytical size exclusion chromatography (data not shown).</p><h3 class="c-article__sub-heading" id="Sec5">Coordination environment of methylcob(III)alamin bound to core-MetE<sub>CBDB</sub> </h3><p>The binding of methylcob(III)alamin to core-MetE<sub>CBDB</sub> was analyzed spectrophotometrically after mixing methylcob(III)alamin and core-MetE<sub>CBDB</sub> in a 1:1 stoichiometry at a concentration of 10 µM each. Free methylcob(III)alamin exhibited α/β- and γ-absorption bands characteristic for the “base-on” mode (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig2">2</a>, violet line)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 38" title="Bandarian, V. & Matthews, R. G. Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. Methods Enzymol. 380, 152–169 (2004)." href="/articles/s41598-020-58873-z#ref-CR38" id="ref-link-section-d466690512e1040">38</a></sup>. On binding to core-MetE<sub>CBDB</sub>, the UV/Vis spectrum of methylcob(III)alamin slightly changed: the absorption intensities of β- and γ-bands increased and the absorption maximum of the β-band blue-shifted slightly (Δλ = −5 nm) (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig2">2</a>, red line).</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-2" data-title="Figure 2"><figure><figcaption><b id="Fig2" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 2</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/2" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig2_HTML.png?as=webp"><img aria-describedby="Fig2" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig2_HTML.png" alt="figure 2" loading="lazy" width="685" height="548"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-2-desc"><p>Coordination mode of methylcob(III)alamin (MeCbl(III)) bound to core-MetE<sub>CBDB</sub> from <i>Dehalococcoides mccartyi</i> strain CBDB1. Free MeCbl(III) in the “base-on” mode is characterized by broad α/β-absorbance bands and characteristic maxima at ~ 487 and 524 nm (violet line). The UV/Vis spectrum of MeCbl(III) in the presence of core-MetE<sub>CBDB</sub> at 1:1 stoichiometry was slightly changed. The absorbance intensities of β- and γ-bands increased and the maximum of the β-band shifted (Δλ = −5 nm) (red line), indicating the “base-off/His-on” binding mode of MeCbl(III).</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/2" data-track-dest="link:Figure2 Full size image" aria-label="Full size image figure 2" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><h3 class="c-article__sub-heading" id="Sec6">Core-MetE<sub>CBDB</sub> catalyzes methionine formation with methylcob(III)alamin as methyl donor</h3><p>The enzymatic activity of purified core-MetE<sub>CBDB</sub> was tested using methylcob(III)alamin as methyl donor and homocysteine as methyl acceptor. In the presence of core-MetE<sub>CBDB</sub>, the UV/Vis absorption spectrum of methylcob(III)alamin, exhibiting a characteristic maximum at 524 nm, successively changed over time due to the consumption of methylcob(III)alamin and formation of cob(I)alamin and cob(II)alamin, as indicated by the emergence of absorption features at 681 nm and 474 nm, respectively (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3a</a>). In the absence of core-MetE<sub>CBDB</sub> or homocysteine, the UV/Vis spectrum of methylcob(III)alamin remained unchanged (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3b,c</a>). In order to exclude any methyltransferase activity due to impurities of the protein preparation, <i>E. coli</i> cell-free extract was also tested and did not show any activity (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3d</a>). Finally, in addition to the photometric measurements, the formation of methionine ([M + H]+ = 150.0583 m/z) during the enzymatic reaction was verified <i>via</i> liquid chromatography-mass spectrometry (LC-MS) (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">2b</a>). In the following, core-MetE<sub>CBDB</sub> enzyme activity was monitored by measuring the increase of absorption at 681 nm (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3e,f</a>) or the decrease of absorption at 524 nm (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">2a</a>). Kinetic parameters for core-MetE<sub>CBDB</sub> were determined using an enzyme concentration of 0.1 µM. At a constant D,L-homocysteine concentration of 2 mM and varying methylcob(III)alamin concentrations, methionine was formed with a <i>V</i><sub>max</sub> = 1664 ± 50 nkat mg<sup>−1</sup> and a <i>K</i><sub>M</sub> = 236 ± 3 µM for methylcob(III)alamin (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3e</a>). When different D,L-homocysteine concentrations were used at a fixed methylcob(III)alamin concentration of 0.5 mM, a <i>V</i><sub>max</sub> = 1582 ± 11 nkat mg<sup>−1</sup> and a <i>K</i><sub>M</sub> = 98 ± 0 µM for D,L-homocysteine were estimated (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3f</a>). Since methionine synthase is specific for L-homocysteine, the apparent <i>K</i><sub>M</sub> for L-homocysteine might be half of that for D,L-homocysteine<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e1154">33</a></sup>. The maximum turnover number (<i>k</i><sub>cat</sub>) was calculated to be about 60 s<sup>−1</sup>. The substrate specificity of core-MetE<sub>CBDB</sub> was investigated by replacing homocysteine with 2 mM cysteine, 2 mM glutathione or 2 mM dithiothreitol. Core-MetE<sub>CBDB</sub> did not show any activity towards these thiol analogs (data not shown).</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-3" data-title="Figure 3"><figure><figcaption><b id="Fig3" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 3</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/3" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig3_HTML.png?as=webp"><img aria-describedby="Fig3" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig3_HTML.png" alt="figure 3" loading="lazy" width="685" height="338"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-3-desc"><p>Demethylation of methylcob(III)alamin (MeCbl(III)) catalyzed by purified core-MetE<sub>CBDB</sub> from <i>D. mccartyi</i> strain CBDB1 in the presence of D,L-homocysteine. (<b>a</b>) In the presence of 0.1 µM core-MetE<sub>CBDB</sub>, 0.5 mM methylcob(III)alamin and 2 mM D,L-homocysteine, continuous changes in the UV/Vis spectrum were observed indicating the consumption of methylcob(III)alamin (524 nm) and the formation of cob(I)alamin (681 nm) and cob(II)alamin (474 nm) as highlighted by arrows. (<b>b</b>) In the absence of core-MetE<sub>CBDB</sub>, the UV/Vis spectrum remained unchanged over an incubation time of 30 min. (<b>c</b>) In the absence of D,L-homocysteine, the UV/Vis spectrum remained unchanged. (<b>d</b>) In the presence of 0.5 mg mL<sup>−1</sup> <i>E. coli</i> crude extract, instead of core-MetE<sub>CBDB</sub> as catalyst, MeCbl(III) was not transformed. (<b>e</b>) Dependence of core-MetE<sub>CBDB</sub> methyltransferase activity on methylcob(III)alamin concentration. (<b>f</b>) Dependence of core-MetE<sub>CBDB</sub> methyltransferase activity on D,L-homocysteine concentration. The activities in panels (<b>e</b>,<b>f</b>) were determined by following the change of the absorption at 681 nm. <i>V</i><sub>max</sub> and <i>K</i><sub>M</sub> were calculated according to a Hill-Fit plot with R<sup>2</sup> = 0.998 for panel (<b>e</b>) and R<sup>2</sup> = 0.999 for panel (<b>f</b>), respectively.</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/3" data-track-dest="link:Figure3 Full size image" aria-label="Full size image figure 3" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>Additionally, 5-methyl-THF-Glu<sub>3</sub> was tested as a methyl group donor for core-MetE<sub>CBDB</sub> instead of methylcobalamin (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig4">4b</a>). In the negative control and also in the presence of core-MetE<sub>CBDB</sub>, slow demethylation of 5 methyl-THF-Glu<sub>3</sub> occurred abiotically<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Steindal, A. H., Juzeniene, A., Johnsson, A. & Moan, J. Photodegradation of 5-methyltetrahydrofolate: biophysical aspects. Photochem. Photobiol. 82, 1651–1655 (2006)." href="#ref-CR39" id="ref-link-section-d466690512e1268">39</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Verlinde, P. H. C. J. et al. Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments. J. Agric. Food Chem. 58, 6190–6199 (2010)." href="#ref-CR40" id="ref-link-section-d466690512e1268_1">40</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 41" title="Liu, Y. et al. Thermal oxidation studies on reduced folate, L-5-methyltetrahydrofolic acid (L-5-MTHF) and strategies for stabilization using food matrices. J. Food Sci. 77, C236–43 (2012)." href="/articles/s41598-020-58873-z#ref-CR41" id="ref-link-section-d466690512e1271">41</a></sup>. The demethylation of 5-methyl-THF-Glu<sub>3</sub> in the negative control and in the presence of core-MetE<sub>CBDB</sub> was not linked to L methionine formation (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig4">4b(I)</a>), while in the presence of tr-MetE<sub>Eco</sub>, methionine was formed exhibiting a signal at [M + H]<sup>+</sup> = 150.0583 m/z (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig4">4b(II)</a>).</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-4" data-title="Figure 4"><figure><figcaption><b id="Fig4" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 4</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/4" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig4_HTML.png?as=webp"><img aria-describedby="Fig4" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig4_HTML.png" alt="figure 4" loading="lazy" width="685" height="559"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-4-desc"><p>Core-MetE<sub>CBDB</sub> from <i>D. mccartyi</i> strain CBDB1 does not catalyze the formation of L-methionine with 5-methyl-THF-Glu<sub>3</sub> as the methyl donor. (<b>a</b>) Reaction described for tr-MetE from <i>E. coli</i> (tr-MetE<sub>Eco</sub>) catalyzing the methylation of L-homocysteine with 5-methyl-THF-Glu<sub>3</sub> to form L-methionine and THF-Glu<sub>3</sub><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 80" title="Eichel, J., González, J. C., Hotze, M., Matthews, R. G. & Schröder, J. Vitamin-B12-Independent Methionine Synthase from a Higher Plant (Catharanthus Roseus). Eur. J. Biochem. 230, 1053–1058 (1995)." href="/articles/s41598-020-58873-z#ref-CR80" id="ref-link-section-d466690512e1319">80</a></sup>. (<b>b</b>) Representative HPLC chromatograms of a chemical standard of 5-methyl-THF-Glu<sub>3</sub> (blue), of reaction products after an enzyme activity assay containing 5-methyl-THF-Glu<sub>3</sub>, D,L-homocysteine and either core-MetE<sub>CBDB</sub> (red) or tr-MetE<sub>Eco</sub> (green) or no enzyme (black). The peak at RT = 21 min represents dithiothreitol added to all reactions. In the presence of tr-MetE<sub>Eco</sub>, 5-methyl-THF-Glu<sub>3</sub> (RT = 18 min) reacts to form THF-Glu<sub>3</sub> (RT = 17.4 min). Slow demethylation of 5-methyl-THF-Glu<sub>3</sub> to THF-Glu<sub>3</sub> did occur in the negative control and also in the presence of core-MetE<sub>CBDB</sub>. To evaluate if this demethylation was linked to L-methionine formation, the products of the activity assays were analyzed by mass spectrometry. I) No L-methionine was formed in the presence of core-MetE<sub>CBDB</sub>; II) The product of tr-MetE<sub>Eco</sub> was identified as L-methionine ([M + H]+ = 150.0583 m/z).</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/4" data-track-dest="link:Figure4 Full size image" aria-label="Full size image figure 4" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><h3 class="c-article__sub-heading" id="Sec7">pH-optimum and thermal stability of core-MetE<sub>CBDB</sub> </h3><p>Methionine synthase activity of core-MetE<sub>CBDB</sub> was observed between pH 5.0 and 9.0, with an optimum between pH 6 and 6.5 (Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-020-58873-z#Tab1">1</a>). The thermal stability of purified tr-MetE<sub>Eco</sub> and core-MetE<sub>CBDB</sub> were assessed by recording protein melting curves using nano differential scanning fluorimetry (nanoDSF). For tr-MetE<sub>Eco</sub>, a melting temperature T<sub>m</sub> = 55.8 ± 0.2 °C was determined. In contrast, the T<sub>m</sub> of core-MetE<sub>CBDB</sub> was at 68.8 ± 0.0 °C (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">3</a>).</p><div class="c-article-table" data-test="inline-table" data-container-section="table" id="table-1"><figure><figcaption class="c-article-table__figcaption"><b id="Tab1" data-test="table-caption">Table 1 Demethylation of methylcob(III)alamin (MeCbl(III)) catalyzed by core-MetE<sub>CBDB</sub> from <i>D. mccartyi</i> strain CBDB1 in the presence of D,L-homocysteine at different pH values.</b></figcaption><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="table-link" data-track="click" data-track-action="view table" data-track-label="button" rel="nofollow" href="/articles/s41598-020-58873-z/tables/1" aria-label="Full size table 1"><span>Full size table</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><h3 class="c-article__sub-heading" id="Sec8">Core-MetE<sub>CBDB</sub> does not complement tr-<i>metE</i>-deficient <i>E. coli in vivo</i> </h3><p>The examination of enzymatic activities of core-MetE<sub>CBDB</sub> <i>in vitro</i> has limitations. However, we were not able to conduct <i>in vivo</i> mutagenesis studies with strain CBDB1 because <i>Dehalococcoide</i>s species are not yet genetically accessible. In order to obtain insights into the physiological role of the cbdbA481 gene product <i>in vivo</i>, we tested whether a tr-<i>metE</i>-deficient <i>E. coli</i> strain could be complemented by core-MetE<sub>CBDB</sub>. Therefore, we generated a tr-<i>metE</i>-deficient knockout strain of <i>E. coli</i> DH5α (<i>ΔmetE::kan</i>) that still contained the <i>metH</i> gene for the cobalamin-dependent MetH. This strain was not able to grow in medium without added cyanocobalamin (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">4</a>, red solid line), but grew when cyanocobalamin was supplemented (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">4</a>, red dotted line). Next, the growth behavior of the mutant strain carrying different complementation plasmids was investigated. Either the original tr-<i>metE</i><sub>Eco</sub> gene or the core-MetE<sub>CBDB</sub> nucleotide sequence, both under the control of an arabinose promotor, were provided. Growth experiments with these complementation strains showed that neither of the two strains grew without inducing gene expression by arabinose. After induction with arabinose, tr-<i>metE</i><sub>Eco</sub> was able to complement the <i>ΔmetE</i> strain as expected (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">4</a>, blue dotted line), while core-MetE<sub>CBDB</sub> was not (Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">4</a>, green dotted line). These results suggested that core-MetE<sub>CBDB</sub> does not have the same physiological function as the canonical tr-MetE<sub>Eco</sub>.</p></div></div></section><section data-title="Discussion"><div class="c-article-section" id="Sec9-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec9">Discussion</h2><div class="c-article-section__content" id="Sec9-content"><p>Methionine and SAM have been suggested to belong to the most ancient molecules on earth and might have emerged within or even before the “RNA world”<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Parker, E. T. et al. Primordial synthesis of amines and amino acids in a 1958 Miller H2S-rich spark discharge experiment. Proc. Natl Acad. Sci. USA 108, 5526–5531 (2011)." href="#ref-CR42" id="ref-link-section-d466690512e1705">42</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Parker, E. T. et al. Prebiotic synthesis of methionine and other sulfur-containing organic compounds on the primitive Earth: a contemporary reassessment based on an unpublished 1958 Stanley Miller experiment. Orig. Life Evol. Biosph. 41, 201–212 (2011)." href="#ref-CR43" id="ref-link-section-d466690512e1705_1">43</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 44" title="Laurino, P. & Tawfik, D. S. Spontaneous emergence of S-adenosylmethionine and the evolution of methylation. Angew. Chem. Int. Ed. 56, 343–345 (2017)." href="/articles/s41598-020-58873-z#ref-CR44" id="ref-link-section-d466690512e1708">44</a></sup>. Although methionine appears to have a continued central metabolic role for more than three billion years, different routes for its biosynthesis have evolved. The biochemically conserved methionine pathway appears to be the product of an evolutionary patchwork involving diverse methionine synthases<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Gophna, U., Bapteste, E., Doolittle, W. F., Biran, D. & Ron, E. Z. Evolutionary plasticity of methionine biosynthesis. Gene 355, 48–57 (2005)." href="/articles/s41598-020-58873-z#ref-CR5" id="ref-link-section-d466690512e1712">5</a></sup>. In our study, we identified a novel bacterial MetE-like methionine synthase in <i>D. mccartyi</i> strain CBDB1 that uses methylcobalamin as methyl donor instead of methylated tetrahydrofolate. Our results suggest that this enzyme is the basal form of canonical tandem-repeat MetE (tr-MetE) proteins with roughly half its size and without the domain duplication of canonical MetE proteins evolved to enable tetrahydrofolate binding on the N-terminal domain<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 19" title="Pejchal, R. & Ludwig, M. L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. PLoS Biol. 3, 254–265 (2005)." href="/articles/s41598-020-58873-z#ref-CR19" id="ref-link-section-d466690512e1719">19</a></sup>. Homologs of this short methionine synthase are encoded in the genomes of several deeply-rooting obligate anaerobic microorganisms from both prokaryotic domains, including all <i>Dehalococcoidia</i> and many <i>Clostridia</i> (<i>e.g. Desulfitobacterium metallireducens, C. kluyveri, C. oryzae</i>) as well as almost all archaea sequenced so far (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>). We refer to this short monomeric MetE form as “core-MetE”, because several lines of evidence hint at its basal descendence including the lack of duplication, the exclusive presence in deeply rooting phylogenetic taxa, and the dependence on corrinoids, which are thought to be ancient cofactors<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 45" title="Decker, K., Jungermann, K. & Thauer, R. K. Energy production in anaerobic organisms. Angew. Chem. Int. Ed. 9, 138–158 (1970)." href="/articles/s41598-020-58873-z#ref-CR45" id="ref-link-section-d466690512e1736">45</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 46" title="Martin, W. F. & Sousa, F. L. Early microbial evolution: the age of anaerobes. Cold Spring Harb. Perspect. Biol. 8, a018127 (2015)." href="/articles/s41598-020-58873-z#ref-CR46" id="ref-link-section-d466690512e1739">46</a></sup>, as they participate in fundamental processes such as ribonucleotide reduction<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 47" title="Dickman, S. R. Ribonucleotide reduction and the possible role of cobalamin in evolution. J. Mol. Evol. 10, 251–260 (1977)." href="/articles/s41598-020-58873-z#ref-CR47" id="ref-link-section-d466690512e1743">47</a></sup>, the Wood-Ljungdahl-pathway<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 48" title="Menon, S. & Ragsdale, S. W. The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein. J. Biol. Chem. 274, 11513–11518 (1999)." href="/articles/s41598-020-58873-z#ref-CR48" id="ref-link-section-d466690512e1747">48</a></sup> and methane formation<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 49" title="Thauer, R. K. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiol. 144, 2377–2406 (1998)." href="/articles/s41598-020-58873-z#ref-CR49" id="ref-link-section-d466690512e1752">49</a></sup>.</p><p>Compared with tr-MetE<sub>Eco</sub>, core-MetE<sub>CBDB</sub> is more stable towards pH changes<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 18" title="Fu, T.-M. et al. Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans. A dynamic zinc-inversion model. J. Mol. Biol. 412, 688–697 (2011)." href="/articles/s41598-020-58873-z#ref-CR18" id="ref-link-section-d466690512e1763">18</a></sup> and thermal denaturation. The turnover number <i>k</i><sub>cat</sub> ≈ 60 s<sup>−1</sup> of core-MetE<sub>CBDB</sub> is very high in comparison to other methionine synthases such as tr-MetE<sub>Eco</sub> with 0.4 s<sup>−1</sup> <sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 50" title="González, J. C., Peariso, K., Penner-Hahn, J. E. & Matthews, R. G. Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme. Biochem. 35, 12228–12234 (1996)." href="/articles/s41598-020-58873-z#ref-CR50" id="ref-link-section-d466690512e1780">50</a></sup> or <i>E. coli</i> MetH with 26 s<sup>−1</sup> <sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 51" title="Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H. & Matthews, R. G. Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme. Biochem. 27, 8458–8465 (1988)." href="/articles/s41598-020-58873-z#ref-CR51" id="ref-link-section-d466690512e1790">51</a></sup>. The activity of MetH is based on domain movements, which could contribute to the lower catalytic rate in comparison to a small monomeric core-MetE. The relatively slow conversion rate of tr-MetE proteins can be due to the poor methylation power of 5-methyl-THF-Glu<sub>n</sub> (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig4">4a</a>) and the weak nucleophilicity of homocysteine at physiological pH<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 52" title="Matthews, R. G. et al. Cobalamin-dependent and cobalamin-independent methionine synthases: are there two solutions to the same chemical problem? Helvetica Chim. Acta 86, 3939–3954 (2003)." href="/articles/s41598-020-58873-z#ref-CR52" id="ref-link-section-d466690512e1799">52</a></sup>. In tr-MetE and MetH, 5-methyl-THF must be activated for the nucleophilic attack by protonation at N<sup>5</sup> <sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Drummond, J. T., Huang, S., Blumenthal, R. M. & Matthews, R. G. Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli. Biochem. 32, 9290–9295 (1993)." href="/articles/s41598-020-58873-z#ref-CR15" id="ref-link-section-d466690512e1805">15</a></sup>. In both MetE and MetH, the nucleophilicity of homocysteine is enhanced by coordination with Zn<sup>2+</sup> that serves as Lewis acid<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1, 332–339 (1997)." href="/articles/s41598-020-58873-z#ref-CR13" id="ref-link-section-d466690512e1812">13</a></sup>. While in the “base-on” form the dimethylbenzimidazole (Dmbz) base of methylcob(III)alamin is coordinated to the cobalt center of the corrin ring, in the “base-off” mode Dmbz is dissociated from the cobalt. Stabilization of the transition state of methylcob(III)alamin in the “base-off” or “base-off/His-on” binding mode enable nucleophilic attack of homocysteine by weakening the Co-C bond and by reducing the thermodynamic barrier<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Martin, B. D. & Finke, R. G. Cobalt-carbon homolysis and bond dissociation energy studies of biological alkylcobalamins: methylcobalamin, including a >1015 Co-CH3 homolysis rate enhancement at 25 °C following one-electron reduction. J. Am. Chem. Soc. 112, 2419–2420 (1990)." href="#ref-CR53" id="ref-link-section-d466690512e1816">53</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" title="Martin, B. D. & Finke, R. G. Methylcobalamin’s full- vs. half-strength cobalt-carbon σ bonds and bond dissociation enthalpies: A > 1015 Co-CH3 homolysis rate enhancement following one-antibonding-electron reduction of methlycobalamin. J. Am. Chem. Soc. 114, 585–592 (1992)." href="#ref-CR54" id="ref-link-section-d466690512e1816_1">54</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 55" title="Jensen, K. P. & Ryde, U. Conversion of homocysteine to methionine by methionine synthase. A density functional study. J. Am. Chem. Soc. 125, 13970–13971 (2003)." href="/articles/s41598-020-58873-z#ref-CR55" id="ref-link-section-d466690512e1819">55</a></sup>. Thus, only binding modes “base-off” or “base-off/His-on” enable methyl transfer from methylcob(III)alamin. However, the “base-off” mode of methylcob(III)alamin which is characterized by strong spectral changes namely, a significant blue shift in the UV/Vis spectrum and reduced intensity of the γ-band<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 38" title="Bandarian, V. & Matthews, R. G. Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. Methods Enzymol. 380, 152–169 (2004)." href="/articles/s41598-020-58873-z#ref-CR38" id="ref-link-section-d466690512e1823">38</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 56" title="Männel-Croisé, C. & Zelder, F. Immobilised vitamin B12 as a biomimetic model for base-off/histidine-on coordination. Chem. Commun. 47, 11249–11251 (2011)." href="/articles/s41598-020-58873-z#ref-CR56" id="ref-link-section-d466690512e1826">56</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 57" title="Ragsdale, S. W., Lindahl, P. A. & Münck, E. Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl-CoA by Clostridium thermoaceticum. J. Biol. Chem. 262, 14289–14297 (1987)." href="/articles/s41598-020-58873-z#ref-CR57" id="ref-link-section-d466690512e1829">57</a></sup>, was not observed in our study (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig2">2</a>). It is difficult to precisely distinguish between the “base-on” and “base-off/His-on” form because, the UV/Vis spectra of them are very similar<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 56" title="Männel-Croisé, C. & Zelder, F. Immobilised vitamin B12 as a biomimetic model for base-off/histidine-on coordination. Chem. Commun. 47, 11249–11251 (2011)." href="/articles/s41598-020-58873-z#ref-CR56" id="ref-link-section-d466690512e1836">56</a></sup>. The formation of methionine and cob(I)alamin (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig3">3a</a>) can only take place if methylcob(III)alamin and homocysteine are bound to core-MetE<sub>CBDB</sub> in a stable and catalytically favorable configuration. Due to the minor spectral changes, we propose that methylcob(III)alamin is utilized by core-MetE<sub>CBDB</sub> in the “base-off/His-on” binding mode. The “base-off/His-on” binding mode is found in many B<sub>12</sub>-dependent proteins with a consensus motif Dx<b>H</b>xxG, where His represents the lower axial ligand replacing the Dmbz moiety<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 58" title="Drennan, C., Huang, S., Drummond, J., Matthews, R. & Lidwig, M. How a protein binds B12. A 3.0 A X-ray structure of B12-binding domains of methionine synthase. Science 266, 1669–1674 (1994)." href="/articles/s41598-020-58873-z#ref-CR58" id="ref-link-section-d466690512e1853">58</a></sup>. In our computational model of core-MetE<sub>CBDB</sub>, His122 points towards the active site of the protein and probably belongs to a truncated B<sub>12</sub>-binding motif with the sequence <b>H</b>xxG, conserved among all <i>Dehalococcoidia</i> (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig5">5</a>).</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-5" data-title="Figure 5"><figure><figcaption><b id="Fig5" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 5</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/5" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig5_HTML.png?as=webp"><img aria-describedby="Fig5" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig5_HTML.png" alt="figure 5" loading="lazy" width="685" height="661"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-5-desc"><p>Computational model of the active site of core-MetE<sub>CBDB</sub> from <i>Dehalococcoides mccartyi</i> strain CBDB1. His122 is tuned towards the active site of the protein where a zinc atom is coordinated by His215, Cys217 and Cys312. His122 might replace the dimethylbenzimidazole moiety of cobalamin in the “bae-off/His-on” mode. The structure was calculated with the I-TASSER server<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 65" title="Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinfomatics 9, 1–8 (2008)." href="/articles/s41598-020-58873-z#ref-CR65" id="ref-link-section-d466690512e1886">65</a></sup> and visualized with PyMOL<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 66" title="DeLano, W. L. PyMol: an open-source molecular graphics tool. CCP4 Newsletter on Protein Crystallography. 40, 82–92 (2002)." href="/articles/s41598-020-58873-z#ref-CR66" id="ref-link-section-d466690512e1890">66</a></sup>.</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/5" data-track-dest="link:Figure5 Full size image" aria-label="Full size image figure 5" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>The determined <i>K</i><sub>M</sub>-value of core-MetE<sub>CBDB</sub> for methylcob(III)alamin of ~ 240 µM is likely much higher than the intracellular concentration of free methylcobalamin<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 29" title="Yan, J., Ritalahti, K. M., Wagner, D. D. & Löffler, F. E. Unexpected specificity of interspecies cobamide transfer from Geobacter spp. to organohalide-respiring Dehalococcoides mccartyi strains. Appl. Env. Microbiol. 78, 6630–6636 (2012)." href="/articles/s41598-020-58873-z#ref-CR29" id="ref-link-section-d466690512e1911">29</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 59" title="Yan, J. et al. The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring Dehalococcoides mccartyi. ISME J. 10, 1092–1101 (2016)." href="/articles/s41598-020-58873-z#ref-CR59" id="ref-link-section-d466690512e1914">59</a></sup>. Therefore, the physiological methyl donor might not be soluble methyl(III)cobalamin. We hypothesize that the physiological methyl donor is a corrinoid protein that directly interacts with core-MetE<sub>CBDB</sub>. Needless to say, that inference of physiological characteristics from the determination of enzyme activity <i>in vitro</i> is limited. Examining the role of core-MetE <i>in vivo</i> could shed more light on the essentiality and functionality of the enzyme. However, genetic modification of <i>Dehalococcoides</i> strains is not possible yet.</p><p>The methyl group transferred by <i>Dehalococcoides</i> methionine synthase origins from exogenously supplied acetate, as has been shown by Zhuang <i>et al</i>.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 32" title="Zhuang, W.-Q. et al. Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi. Proc. Natl Acad. Sci. USA 111, 6419–6424 (2014)." href="/articles/s41598-020-58873-z#ref-CR32" id="ref-link-section-d466690512e1939">32</a></sup>. Acetate is activated in <i>Dehalococcoides</i> by acetyl-CoA synthetase (ACS) to acetyl-CoA<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 31" title="Marco-Urrea, E., Seifert, J., Bergen, Mvon & Adrian, L. Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1. J. Bacteriol. 194, 4169–4177 (2012)." href="/articles/s41598-020-58873-z#ref-CR31" id="ref-link-section-d466690512e1946">31</a></sup>. Acetyl-CoA is then cleaved to free coenzyme A, carbon monoxide (which leaves the cell) and a methyl group originating from the C<sub>2</sub>-atom of acetate. This reaction is catalyzed by acetyl-CoA decarbonylase/synthase (AcsB), an enzyme known mostly for its activity in the opposite direction for carbon fixation <i>via</i> the Wood-Ljungdahl pathway<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 60" title="Ljungdahl, L. G. & Wood, H. G. Total synthesis of acetate from CO2 by heterotrophic bacteria. Annu. Rev. microbiology 23, 515–538 (1969)." href="/articles/s41598-020-58873-z#ref-CR60" id="ref-link-section-d466690512e1956">60</a></sup>. In <i>D. mccartyi</i> strain CBDB1, AcsB represents the acetyl-CoA decarbonylase and AcsCD a dimeric corrinoid iron-sulfur protein (CoFeSP) to which the methyl group from acetyl-CoA is transferred<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 61" title="Matthews, R. G., Koutmos, M. & Datta, S. Cobalamin-dependent and cobamide-dependent methyltransferases. Curr. Opin. Struct. Biol. 18, 658–666 (2008)." href="/articles/s41598-020-58873-z#ref-CR61" id="ref-link-section-d466690512e1963">61</a></sup>. Zhuang <i>et al</i>. hypothesized that the methyl group is then transferred from AcsCD to tetrahydrofolate and from there to homocysteine, but <i>Dehalococcoides</i> neither encode the methyltransferases <i>acsE</i>, responsible for the methyl transfer from CoFeSP to tetrahydrofolate nor the classical <i>metE</i>/<i>metH</i>, responsible for methyl transfer from methyl-tetrahydrofolate to homocysteine (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig6">6a,b</a>)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 32" title="Zhuang, W.-Q. et al. Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi. Proc. Natl Acad. Sci. USA 111, 6419–6424 (2014)." href="/articles/s41598-020-58873-z#ref-CR32" id="ref-link-section-d466690512e1986">32</a></sup>. Our results can now explain these two gaps by hypothesizing that the methyl group from AcsCD/CoFeSP is directly transferred to homocysteine by core-MetE<sub>CBDB</sub> instead of taking the diversion <i>via</i> tetrahydrofolate (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig6">6c</a>). This hypothesis would also explain the absence of carbon monoxide dehydrogenase in <i>Dehalococcoides</i> which would be needed if the Wood-Ljungdahl pathway was employed for CO<sub>2</sub> fixation. With the direct transfer of methyl groups from CoFeSP to homocysteine, the cells would be independent from methyl-tetrahydrofolate and indeed <i>metF</i> encoding methylene-tetrahydrofolate reductase (MTHFR) is missing in all <i>Dehalococcoides</i> genomes<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 27" title="Seshadri, R. et al. Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes. Sci. 307, 105–108 (2005)." href="/articles/s41598-020-58873-z#ref-CR27" id="ref-link-section-d466690512e2011">27</a></sup>. This might be an unusual pathway in extant microbiology but in our view could represent a very early evolutionary stage in which methyl metabolism could have been independent from folates. This view is supported by the fact that methionine, SAM, corrinoids and coenzyme A are conserved between archaea and bacteria but tetrahydrofolate/tetrahydromethanopterin are not<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 45" title="Decker, K., Jungermann, K. & Thauer, R. K. Energy production in anaerobic organisms. Angew. Chem. Int. Ed. 9, 138–158 (1970)." href="/articles/s41598-020-58873-z#ref-CR45" id="ref-link-section-d466690512e2015">45</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 46" title="Martin, W. F. & Sousa, F. L. Early microbial evolution: the age of anaerobes. Cold Spring Harb. Perspect. Biol. 8, a018127 (2015)." href="/articles/s41598-020-58873-z#ref-CR46" id="ref-link-section-d466690512e2018">46</a></sup>.</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-6" data-title="Figure 6"><figure><figcaption><b id="Fig6" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 6</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/6" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig6_HTML.png?as=webp"><img aria-describedby="Fig6" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_Fig6_HTML.png" alt="figure 6" loading="lazy" width="685" height="645"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-6-desc"><p>Pathways of L-glycine and L-homocysteine methylation in <i>Dehalococcoides</i> species and hypothesized involvement of THF and corrinoid proteins. (<b>a</b>) Genes annotated to be involved in the incomplete Wood-Ljungdahl pathway encoded in <i>D. mccartyi</i> strain 195 and the respective homologous genes in other <i>Dehalococcoidia</i>. (<b>b</b>) L-serine formation <i>via</i> glycine hydroxymethyltransferase (GlyA, grey) as proposed by Zhuang <i>et al</i>. is shown<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 32" title="Zhuang, W.-Q. et al. Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi. Proc. Natl Acad. Sci. USA 111, 6419–6424 (2014)." href="/articles/s41598-020-58873-z#ref-CR32" id="ref-link-section-d466690512e2054">32</a></sup>. The methyl group is derived most probably from formate with the aid of formyl-tetrahydrofolate synthase (Fhs, yellow) and methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD, blue). (<b>c</b>) Methylation of L-homocysteine is conducted by core-MetE<sub>CBDB</sub> encoded by the locus cbdbA481 (purple) in <i>D. mccartyi</i> strain CBDB1. The original source of the methyl group is acetate, which is activated to acetyl-CoA and then cleaved by acetyl-CoA decarbonylase (AcsB, green) into HSCoA, carbon monoxide (CO) and a methyl group. The standard activity of AcsB is to transfer the methyl group to a corrinoid iron-sulfur protein complex (CoFeSP) AcsCD (red). We speculate that the methyl group is directly transferred from the CoFeSP to the core-MetE<sub>CBDB</sub> (A481) for L-homocysteine methylation (dashed arrow) but this transfer could also be indirect <i>via</i> a yet unidentified participant.</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/s41598-020-58873-z/figures/6" data-track-dest="link:Figure6 Full size image" aria-label="Full size image figure 6" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>Enzymes similar to the core-MetE identified in <i>Dehalococcoides</i> were also found in the majority of archaea (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>, blue colors). <i>M. thermoautotrophicum</i> and other methanogens are described to encode methylcobalamin:homocysteine methyltransferase (core-MetE<sub>Archaea</sub>)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e2095">33</a></sup>, a protein of 308 amino acids that is also homologous to the C-terminal part of tr-MetE proteins. <i>In vitro</i> experiments showed, that core-MetE<sub>Archaea</sub> utilizes methylated corrinoids for the methylation of homocysteine, similar to what we now found for the core-MetE<sub>CBDB</sub>. Schröder and Thauer concluded that soluble methylcobalamin is unlikely the physiological methyl group donor and hypothesized that a corrinoid protein with yet unknown function could play this role. The gene products of <i>MTH124</i> or <i>MTH1156</i> were proposed as possible candidates<i>. MTH1156</i> encodes MtrH, a protein with sequence similarity to the 5-methyl-THF-Glu-binding domain of MetH (26% identity)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e2117">33</a></sup>. MtrH is part of the methyl-tetrahydromethanopterin-coenzyme M methyltransferase complex and catalyzes the methylation of cob(I)alamin to methylcob(III)alamin using methyl-tetrahydromethanopterin as methyl group donor<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 62" title="Hippler, B. & Thauer, R. K. The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH. FEBS Lett. 449, 165–168 (1999)." href="/articles/s41598-020-58873-z#ref-CR62" id="ref-link-section-d466690512e2121">62</a></sup>. In contrast to methyl-THF biosynthesis in <i>Dehalococcoides</i> strains, methanopterin biosynthesis, a functional equivalent to THF in archaea, is fully encoded in all methanogenic archaea<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 49" title="Thauer, R. K. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiol. 144, 2377–2406 (1998)." href="/articles/s41598-020-58873-z#ref-CR49" id="ref-link-section-d466690512e2128">49</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 63" title="Ferry, J. G. Fundamentals of methanogenic pathways that are key to the biomethanation of complex biomass. Curr. Opin. Biotechnol. 22, 351–357 (2011)." href="/articles/s41598-020-58873-z#ref-CR63" id="ref-link-section-d466690512e2131">63</a></sup>. The methyl group of methionine in methanogenic archaea is derived from methyl-tetrahydromethanopterin<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 49" title="Thauer, R. K. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiol. 144, 2377–2406 (1998)." href="/articles/s41598-020-58873-z#ref-CR49" id="ref-link-section-d466690512e2135">49</a>,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 64" title="Länge, S. & Fuchs, G. Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum. Eur. J. Biochem. 163, 147–154 (1987)." href="/articles/s41598-020-58873-z#ref-CR64" id="ref-link-section-d466690512e2138">64</a></sup>, which might be the primary methyl donor of archaeal methylcobalamin:homocysteine methyltransferases.</p><p>In conclusion, our findings show that bacterial core-MetE<sub>CBDB</sub> homologs together with archaeal core-MetE representatives form a basal group of methionine synthases using methylcobalamin <i>in vitro</i> as co-substrate. Due to the fact that organisms encoding core-MetE enzymes are slowly growing strict anaerobes with strongly conserved ancient traits, we speculate that the core-MetE homologs are similar to an ancient methionine synthase encoded already in the genome of a predecessor of LUCA and therefore basal to both archaea and bacteria. We speculate that such basal methionine synthases were active in the metabolism of ancient microorganisms using methylcobalamin-containing proteins as methyl donor. Core-MetE<sub>CBDB</sub> is the first biochemically described bacterial representative of these core-MetE proteins resembling the methylcobalamin:homocysteine methyltransferase from <i>M. thermoautotrophicum</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 263, 789–796 (1999)." href="/articles/s41598-020-58873-z#ref-CR33" id="ref-link-section-d466690512e2154">33</a></sup>. Tr-MetE proteins appear to have evolved by duplications of core-MetE and subsequently acquired the capacity to bind folate at the N-terminal part. In our phylogenetic analysis tr-MetE clusters with archaeal core-MetE genes (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig1">1a</a>).</p></div></div></section><section data-title="Materials and Methods"><div class="c-article-section" id="Sec10-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec10">Materials and Methods</h2><div class="c-article-section__content" id="Sec10-content"><h3 class="c-article__sub-heading" id="Sec11">General</h3><p>All chemicals were purchased from Sigma-Aldrich (Munich, Germany) or Carl Roth (Karlsruhe, Germany). Whenever methionine or homocysteine are mentioned in the text, the L-form is meant. Chemicals used for mass spectrometry were obtained in LC-MS grade from Carl Roth. Pteroyltri-γ-L-glutamic acid (PteGlu<sub>3</sub>) was acquired from Schircks Laboratories (Jona, Switzerland). Restriction enzymes, DNA polymerase, DNA and protein standards were obtained from New England BioLabs (Frankfurt/Main, Germany). Oligonucleotides and sequencing services were provided by Seqlab (Göttingen, Germany). All oligonucleotide primers, plasmids and strains used in this study are listed in Supplementary Tables <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">1</a> and <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">2</a>. Anaerobic experiments were performed in a COY glovebox (Grass Lake, USA).</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec12">Bioinformatics</h3><p>The structural model of CbdbA481 (core-MetE<sub>CBDB</sub>) was calculated using the I-TASSER server<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 65" title="Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinfomatics 9, 1–8 (2008)." href="/articles/s41598-020-58873-z#ref-CR65" id="ref-link-section-d466690512e2191">65</a></sup>. Broadly defined, the server aligns the template protein with proteins of similar folds or with super-secondary structures from the PDB library by LOMETS. The overlay of core-MetE<sub>CBDB</sub> and tr-MetE from <i>N. crassa</i> was generated with PyMOL<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 66" title="DeLano, W. L. PyMol: an open-source molecular graphics tool. CCP4 Newsletter on Protein Crystallography. 40, 82–92 (2002)." href="/articles/s41598-020-58873-z#ref-CR66" id="ref-link-section-d466690512e2200">66</a></sup>. The amino acid sequences of tr-MetEs were trimmed approximately at the position 370. For the multiple sequence alignment and construction of the phylogenetic tree, only the C-termini of truncated tr-MetEs from bacteria, yeast and complete amino acid sequences of core-MetEs from archaea, <i>Chloroflexi</i> and <i>Clostridiales</i> were used. MEGA7<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 67" title="Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016)." href="/articles/s41598-020-58873-z#ref-CR67" id="ref-link-section-d466690512e2211">67</a></sup> was used to calculate multiple amino acid sequence alignments using the implemented MUSCLE algorithm with default settings<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 68" title="Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)." href="/articles/s41598-020-58873-z#ref-CR68" id="ref-link-section-d466690512e2215">68</a></sup>. The evolutionary relationship between different methionine synthase amino acid sequences was inferred by using the Maximum Likelihood method based on the JTT matrix model<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 69" title="Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992)." href="/articles/s41598-020-58873-z#ref-CR69" id="ref-link-section-d466690512e2219">69</a></sup>. Evolutionary distances were computed using Poisson correction and are expressed as the number of amino acid substitutions per site<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 70" title="Zuckerkandl, E. & Pauling, L. In Evolving genes and proteins (Elsevier), pp. 97–166 (1965)." href="/articles/s41598-020-58873-z#ref-CR70" id="ref-link-section-d466690512e2223">70</a></sup>.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec13">Construction of expression and complementation plasmids</h3><p>Based on pBAD30, expression and complementation plasmids were generated as described in supplementary information. The resulting plasmids pBAD_MetE and pBAD_CbdbA481 were used for the complementation experiments as well as for the heterologous production and purification of tandem-repeat MetE (tr-MetE<sub>Eco</sub>) from <i>E. coli</i> and core-MetE<sub>CBDB</sub> from <i>D. mccartyi</i> strain CBDB1.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec14">Production and purification of recombinant tr-MetE<sub>Eco</sub> and core-MetE<sub>CBDB</sub> </h3><p>A preculture of <i>E. coli</i> DH10B containing pBAD30_MetE or pBAD30_CbdbA481 was set up in Luria-Bertani (LB) medium containing 100 µg mL<sup>−1</sup> ampicillin and grown overnight at 37 °C and 140 rpm. On the following day, 1% (v/v) of the overnight culture was used to inoculate fresh LB medium containing the appropriate antibiotic. The cultures were grown at 37 °C under agitation at 140 rpm until the OD<sub>600</sub> reached 0.4–0.5. Then, the production of either tr-MetE<sub>Eco</sub> or core-MetE<sub>CBDB</sub> was induced by the addition of 0.05% (w/v) L-arabinose. Additionally, the medium was supplemented with 1 mM ZnSO<sub>4</sub>. MetE and CbdbA481 were produced for 5 h at 37 °C and 140 rpm. Then, the cells were harvested by centrifugation and washed with 50 mM Tris/HCl, pH 7.5. Purification of tr-MetE<sub>Eco</sub> and core-MetE<sub>CBDB</sub> was performed under anoxic conditions in an anaerobic chamber. Both enzymes were purified by anion exchange chromatography using a MonoQ 5/50 GL column connected to an ÄKTA purifier FPLC system (GE Healthcare Life Sciences) as described in detail in the supplementary information.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec15">SDS-PAGE and native PAGE</h3><p>The purity of core-MetE<sub>CBDB</sub> and tr-MetE<sub>Eco</sub> protein preparations was evaluated by 10% SDS-PAGE. In addition, the oligomeric state of both proteins was investigated <i>via</i> 10% discontinuous native PAGE<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 71" title="Ornstein, L. & Davis, B. J. Disc electrophoresis-I: background and theory. Ann. NY. Acad. Sci. 121, 321–349 (1964)." href="/articles/s41598-020-58873-z#ref-CR71" id="ref-link-section-d466690512e2292">71</a></sup>.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec16">Protein identification from SDS-PAGE by LC-MS/MS</h3><p>Qualitative identification of the purified core-MetE<sub>CBDB</sub> and tr-MetE<sub>Eco</sub> proteins was conducted mass spectrometrically. Therefore, protein bands at the height of 38 kDa and 80 kDa were excised from 10% SDS-PAGE gels. Acetonitrile, 10 mM DTT and 100 mM iodoacetamide were used to destain, to reduce and to alkylate the proteins within the gel slices. Subsequently, the proteins were digested with 0.1 µg trypsin (Promega) at 37 °C for 18 h. The resulting peptides were extracted from the gel matrix with 50% (v/v) acetonitrile and 5% (v/v) formic acid and dried. The peptides were again dissolved in 10 µL 0.1% formic acid and subsequently desalted using C<sub>18</sub> ZipTip Pipette Tips (Merck Millipore) and dried in a vacuum centrifuge. Prior analysis, the peptides were resuspended in 20 µL 0.1% formic acid. Samples were analyzed on an LC-MS/MS system composed of a nano-UPLC system (UltiMate 3000 RSLCnano System, Thermo Fisher Scientific) equipped with an Acclaim PepMap 100 75 µm × 25 cm C<sub>18</sub> column and connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) <i>via</i> an electrospray ion source (TriVersa NanoMate, Advion). Sample volumes of 5 µL were injected onto the column and separated applying a flow rate of 0.3 µL min<sup>−1</sup> with the aid of a 60 min gradient from 3.2% to 44% acetonitrile in water containing 0.1% formic acid. The mass spectrometer was operated in positive-ionization mode. The spray voltage was set at 2.2 kV and an electron spray ionization source temperature at 220 °C. Full MS1 scans were obtained over a mass range of 300–2000 m/z and the resolution in the Orbitrap was set to 240,000. The most intense ions (threshold ion count above 5.0 × 10<sup>4</sup>) were selected for fragmentation with the quadrupole, setting the isolation window to 1.6 m/z. Ions were fragmented by ETciD (ETD reaction time 100 ms, CID collision energy 35%). The resulting fragment ion spectra were obtained achieved in the Orbitrap at a resolution of 60,000 and a maximum injection time of 120 ms.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec17">Protein and peptide identification</h3><p>The raw mass spectrometric data were converted to mgf-files using ProteoWizard MSConvert v3.0<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 72" title="Holman, J. D., Tabb, D. L. & Mallick, P. Employing ProteoWizard to convert raw mass spectrometry data. Curr. Protoc. Bioinforma. 46, 13.24.1–9 (2014)." href="/articles/s41598-020-58873-z#ref-CR72" id="ref-link-section-d466690512e2328">72</a></sup>. The software SearchGUI (v3.3.5)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 73" title="Vaudel, M., Barsnes, H., Berven, F. S., Sickmann, A. & Martens, L. SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteom. 11, 996–999 (2011)." href="/articles/s41598-020-58873-z#ref-CR73" id="ref-link-section-d466690512e2332">73</a></sup> and the OMSSA search algorithm were used for peptide identification. Mass spectrometric data were searched against the <i>E. coli</i> proteome database obtained from UniProt (Taxon identifier 316385). A precursor ion mass tolerance of 10 ppm was used at the MS1 level and up to two missed cleavages were allowed. The fragment ion mass tolerance was set to 0.2 Da for the Orbitrap MS2 detection. The oxidation of methionine was considered as variable modification and carbamidomethylation on cysteines as fixed modification. The false discovery rate (FDR) in peptide identification was limited to a maximum of 0.01 by using a decoy database. The analyzed data were visualized with the PeptideShaker software (v1.16.27) (CompOmics, Ghent University)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 74" title="Vaudel, M. et al. PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nat. Biotechnol. 33, 22–24 (2015)." href="/articles/s41598-020-58873-z#ref-CR74" id="ref-link-section-d466690512e2339">74</a></sup>.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec18">Photometric analysis of methylcob(III)alamin binding to core-MetE<sub>CBDB</sub> </h3><p>The binding of methylcob(III)alamin to core-MetE was followed spectrophotometrically in the range between 250 and 800 nm (0.5 nm steps) in 50 mM Tris/HCl (pH 6.5), 150 mM NaCl and 10% glycerol. First, the UV/Vis spectrum of 10 µM free methylcob(III)alamin was recorded. To assess the binding mode of methylcob(III)alamin to core-MetE<sub>CBDB</sub>, the protein solution was mixed with methylcob(III)alamin in a 1:1 stoichiometry (10 µM each). The UV/Vis spectra of free and bound methylcob(III)alamin were compared.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec19">Enzyme activity assay with methylcob(III)alamin as methyl group donor</h3><p>Enzyme activity assays were set up at dim light under strictly anoxic conditions. The standard enzyme assay mix contained 50 mM Tris/HCl (pH 6.5), 150 mM NaCl, 10% glycerol, 0.5 mM methylcob(III)alamin and 0.1 µM enzyme. After 5 min preincubation at room temperature, the reaction was started by the addition of 2 mM D,L-homocysteine. The reaction was photometrically monitored either at 524 nm indicating the consumption of methylcob(III)alamin (ε<sub>524</sub> = 6,200 M<sup>−1</sup> cm<sup>−1</sup>) or at 681 nm indicating the formation of cob(I)alamin (ε<sub>681</sub> = 1,200 M<sup>−1</sup> cm<sup>−1</sup>) (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-58873-z#Fig2">2a</a> and Supplementary Figure <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-020-58873-z#MOESM1">2</a>)<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 75" title="Sauer, K. & Thauer, R. K. Methanol:coenzyme M methyltransferase from Methanosarcina barkeri - substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin. Eur. J. Biochem. 261, 674–681 (1999)." href="/articles/s41598-020-58873-z#ref-CR75" id="ref-link-section-d466690512e2383">75</a></sup>. Enzyme kinetics of core-MetE<sub>CBDB</sub> were performed at concentrations of either 0.5 mM methylcob(III)alamin or 2 mM D,L-homocysteine while the concentration of the second substrate was varied.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec20">Synthesis of (6R,S)-5-methyl-5,6,7,8-tetrahydropteroyltri-γ-L-glutamic acid (5-methyl-THF-Glu<sub>3</sub>)</h3><p>The synthesis of 5-methyl-THF-Glu<sub>3</sub> was accomplished from commercially available PteGlu<sub>3</sub> under anoxic conditions following the modified protocol of Yeo and Wagner<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 76" title="Yeo, E. J. & Wagner, C. Purification and properties of pancreatic glycine N-methyltransferase. J. Biol. Chem. 267, 24669–24674 (1992)." href="/articles/s41598-020-58873-z#ref-CR76" id="ref-link-section-d466690512e2404">76</a></sup> and as described in detail in the supplementary information. 5-methyl-H<sub>4</sub>PteGlu<sub>3</sub> was stored at −20 °C.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec21">Enzyme activity assay with 5-methyl-THF-Glu<sub>3</sub> as methyl group donor</h3><p>Enzyme assays were performed under strictly anoxic conditions. The standard assay was set up in 25 mM Tris/HCl (pH 7.2) or 50 mM KH<sub>2</sub>PO<sub>4</sub>/K<sub>2</sub>HPO<sub>4</sub> (pH 7.2), 100 µM MgSO<sub>4</sub>, 100 µM ZnSO<sub>4,</sub> 10 mM dithiothreitol (DTT), 2 mM D,L-homocysteine and 150 µM 5-methyl-THF-Glu<sub>3</sub>. The reaction was started by the addition of 0.25 µM tr-MetE<sub>Eco</sub> or core-MetE<sub>CBDB</sub>. After an incubation time of 60 min at 37 °C, the reactions were stopped by heat denaturation at 80 °C for 10 min, then centrifuged at 15,000 rpm for 5 min (Eppendorf Centrifuge 5424 R) and analyzed by HPLC. A negative control under same conditions without protein was run to evaluate abiotic transformation of 5-methyl-THF-Glu<sub>3</sub>. 5-methyl-THF-Glu<sub>3</sub>, other folate derivatives and PteGlu<sub>3</sub> were analyzed with a JASCO HPLC 2000 series system equipped with an Equisil BDS C<sub>18</sub> column (250 × 4.6 mm, 5 μm; Dr. Maisch HPLC GmbH, Ammerbuch-Entringen/Germany) following the modified protocol of Patring<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 77" title="Patring, J. D. M., Jastrebova, J. A., Hjortmo, S. B., Andlid, T. A. & Jägerstad, I. M. Development of a simplified method for the determination of folates in baker’s yeast by HPLC with ultraviolet and fluorescence detection. J. Agric. Food Chem. 53, 2406–2411 (2005)." href="/articles/s41598-020-58873-z#ref-CR77" id="ref-link-section-d466690512e2452">77</a></sup>. The identities of PteGlu<sub>3</sub>, 5-methyl-THF-Glu<sub>3</sub> and L-methionine were confirmed <i>via</i> liquid chromatography-mass spectrometry in direct injection mode.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec22">Generation of <i>E. coli</i> knockout strain</h3><p>The <i>E. coli</i> DH5α (Δ<i>metE::kan)</i> knockout strain was generated using the Quick & Easy <i>E. coli</i> Gene Deletion Kit (GeneBridges GmbH, Heidelberg, Germany) according to the manufacturer’s protocol<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 78" title="Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Sys Biol. 2, 1–11 (2006)." href="/articles/s41598-020-58873-z#ref-CR78" id="ref-link-section-d466690512e2484">78</a></sup>. Hereby, <i>metE</i> gene in <i>E. coli</i> DH5α was replaced by a linear kanamycin cassette. The introduction of the kanamycin cassette allowed to screen for the knockout strain on 20 µg mL<sup>−1</sup> kanamycin agar plates.</p><h3 class="c-article__sub-heading c-article__sub-heading--divider" id="Sec23"><i>In vivo</i> complementation of <i>E. coli</i> DH5α (<i>ΔmetE::kan</i>) and cultivation procedure</h3><p>The <i>metE-</i>deficient <i>E. coli</i> DH5α (<i>E. coli</i> DH5α (<i>ΔmetE::kan</i>)) strain was transformed with pBAD30 (negative control), pBAD30_MetE (positive control) or pBAD30_CbdbA481. The first subculture was grown in 5 mL LB medium with 100 µg mL<sup>−1</sup> ampicillin and 20 µg mL<sup>−1</sup> kanamycin at 37 °C and 140 rpm overnight. An inoculum of 1% (v/v) of the first subculture was then used to inoculate the second subculture of 5 mL M9 minimal medium<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 79" title="Harwood, C. R. & Cutting, S. M. In Molecular biological methods in Bacillus, edited by C. R. Harwood & S. M. Cutting (Wiley, Chichester), Vol. 1, p. 548 (1990)." href="/articles/s41598-020-58873-z#ref-CR79" id="ref-link-section-d466690512e2531">79</a></sup> supplemented with 1 mM MgSO<sub>4</sub>, 0.1 mM CaCl<sub>2</sub>, 10 µM FeCl<sub>3</sub>/EDTA, 1.2 mM thiamine, 0.3 mM L-leucine, 0.4% (v/v) glycerol and 0.4 µM cyanocobalamin, that was grown at 37 °C and 140 rpm overnight. Then, several 10 mL-tubes of fresh M9 medium containing all supplements except cyanocobalamin were inoculated with 1% (v/v) of the second subculture. The following main cultures were set up:</p><ol class="u-list-style-none"> <li> <span class="u-custom-list-number">(a)</span> <p><i>E. coli</i> DH5α wild type with or without 0.4 µM cyanocobalamin,</p> </li> <li> <span class="u-custom-list-number">(b)</span> <p><i>E. coli</i> DH5α (<i>ΔmetE::kan</i>) with or without 0.4 µM cyanocobalamin,</p> </li> <li> <span class="u-custom-list-number">(c)</span> <p><i>E. coli</i> DH5α (<i>ΔmetE::kan</i>) + pBAD30_MetE with or without 0.4 µM cyanocobalamin and with or without 0.05% (w/v) L-arabinose,</p> </li> <li> <span class="u-custom-list-number">(d)</span> <p><i>E. coli</i> DH5α (<i>ΔmetE::kan</i>) + pBAD_CbdbA481 with or without 0.4 µM cyanocobalamin and with or without 0.05% (w/v) L-arabinose.</p> </li> </ol><p>The main cultures were then incubated at 37 °C and 140 rpm and growth was monitored by measuring the OD<sub>600</sub>. <i>E. coli</i> DH5α still encodes the arabinose operon. However, arabinose at a concentration of 0.05% (w/v) sufficed to induce the production of core-MetE<sub>CBDB</sub> and tr-MetE<sub>Eco</sub>. In our experiments, reciprocal metabolism leads to preferential use of glycerol instead of arabinose.</p></div></div></section> </div> <div class="u-mt-32"> <div id="MagazineFulltextArticleBodySuffix"><section aria-labelledby="Bib1" data-title="References"><div class="c-article-section" id="Bib1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Bib1">References</h2><div class="c-article-section__content" id="Bib1-content"><div data-container-section="references"><ol class="c-article-references" data-track-component="outbound reference" data-track-context="references section"><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="1."><p class="c-article-references__text" id="ref-CR1">Kozak, M. Initiation of translation in prokaryotes and eukaryotes. <i>Gene</i> <b>234</b>, 187–208 (1999).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/S0378-1119(99)00210-3" data-track-item_id="10.1016/S0378-1119(99)00210-3" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2FS0378-1119%2899%2900210-3" aria-label="Article reference 1" data-doi="10.1016/S0378-1119(99)00210-3">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1MXks1Cqsbg%3D" aria-label="CAS reference 1">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10395892" aria-label="PubMed reference 1">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 1" href="http://scholar.google.com/scholar_lookup?&title=Initiation%20of%20translation%20in%20prokaryotes%20and%20eukaryotes&journal=Gene&doi=10.1016%2FS0378-1119%2899%2900210-3&volume=234&pages=187-208&publication_year=1999&author=Kozak%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="2."><p class="c-article-references__text" id="ref-CR2">Stipanuk, M. H. Metabolism of sulfur-containing amino acids. <i>Ann. Rev. Nutr.</i> <b>6</b>, 179–209 (1986).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1146/annurev.nu.06.070186.001143" data-track-item_id="10.1146/annurev.nu.06.070186.001143" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1146%2Fannurev.nu.06.070186.001143" aria-label="Article reference 2" data-doi="10.1146/annurev.nu.06.070186.001143">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaL28XlsFShtr8%3D" aria-label="CAS reference 2">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 2" href="http://scholar.google.com/scholar_lookup?&title=Metabolism%20of%20sulfur-containing%20amino%20acids&journal=Ann.%20Rev.%20Nutr.&doi=10.1146%2Fannurev.nu.06.070186.001143&volume=6&pages=179-209&publication_year=1986&author=Stipanuk%2CMH"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="3."><p class="c-article-references__text" id="ref-CR3">Stipanuk, M. H. & Ueki, I. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur. <i>J. Inherit. Metab. Dis.</i> <b>34</b>, 17–32 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="noopener" data-track-label="10.1007/s10545-009-9006-9" data-track-item_id="10.1007/s10545-009-9006-9" data-track-value="article reference" data-track-action="article reference" href="https://link.springer.com/doi/10.1007/s10545-009-9006-9" aria-label="Article reference 3" data-doi="10.1007/s10545-009-9006-9">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXhtVKmtbc%3D" aria-label="CAS reference 3">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=20162368" aria-label="PubMed reference 3">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 3" href="http://scholar.google.com/scholar_lookup?&title=Dealing%20with%20methionine%2Fhomocysteine%20sulfur%3A%20cysteine%20metabolism%20to%20taurine%20and%20inorganic%20sulfur&journal=J.%20Inherit.%20Metab.%20Dis.&doi=10.1007%2Fs10545-009-9006-9&volume=34&pages=17-32&publication_year=2011&author=Stipanuk%2CMH&author=Ueki%2CI"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="4."><p class="c-article-references__text" id="ref-CR4">Cantoni, G. L. <i>S</i>-adenosylmethionine; a new intermediate formed enzymatically from L-methionine and adenosinetriphosphate. <i>J. Biol. Chem.</i> <b>204</b>, 403–416 (1953).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaG3sXntlWhtA%3D%3D" aria-label="CAS reference 4">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=13084611" aria-label="PubMed reference 4">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 4" href="http://scholar.google.com/scholar_lookup?&title=S-adenosylmethionine%3B%20a%20new%20intermediate%20formed%20enzymatically%20from%20L-methionine%20and%20adenosinetriphosphate&journal=J.%20Biol.%20Chem.&volume=204&pages=403-416&publication_year=1953&author=Cantoni%2CGL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="5."><p class="c-article-references__text" id="ref-CR5">Gophna, U., Bapteste, E., Doolittle, W. F., Biran, D. & Ron, E. Z. Evolutionary plasticity of methionine biosynthesis. <i>Gene</i> <b>355</b>, 48–57 (2005).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/j.gene.2005.05.028" data-track-item_id="10.1016/j.gene.2005.05.028" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2Fj.gene.2005.05.028" aria-label="Article reference 5" data-doi="10.1016/j.gene.2005.05.028">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2MXntlSgsro%3D" aria-label="CAS reference 5">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=16046084" aria-label="PubMed reference 5">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 5" href="http://scholar.google.com/scholar_lookup?&title=Evolutionary%20plasticity%20of%20methionine%20biosynthesis&journal=Gene&doi=10.1016%2Fj.gene.2005.05.028&volume=355&pages=48-57&publication_year=2005&author=Gophna%2CU&author=Bapteste%2CE&author=Doolittle%2CWF&author=Biran%2CD&author=Ron%2CEZ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="6."><p class="c-article-references__text" id="ref-CR6">Frey, P. A., Ballinger, M. D. & Reed, G. H. <i>S</i>-adenosylmethionine. A ‘poor man’s coenzyme B<sub>12</sub>’ in the reaction of lysine 2,3-aminomutase. <i>Biochm Soc. Trans.</i> <b>26</b>, 304–310 (1998).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1042/bst0260304" data-track-item_id="10.1042/bst0260304" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1042%2Fbst0260304" aria-label="Article reference 6" data-doi="10.1042/bst0260304">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1cXlvVynsb8%3D" aria-label="CAS reference 6">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 6" href="http://scholar.google.com/scholar_lookup?&title=S-adenosylmethionine.%20A%20%E2%80%98poor%20man%E2%80%99s%20coenzyme%20B12%E2%80%99%20in%20the%20reaction%20of%20lysine%202%2C3-aminomutase&journal=Biochm%20Soc.%20Trans.&doi=10.1042%2Fbst0260304&volume=26&pages=304-310&publication_year=1998&author=Frey%2CPA&author=Ballinger%2CMD&author=Reed%2CGH"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="7."><p class="c-article-references__text" id="ref-CR7">Foster, M. A., Tejerina, G., Guest, J. R. & Woods, D. D. Two enzymic mechanisms for the methylation of homocysteine by extracts of <i>Escherichia coli</i>. <i>Biochem.</i> <b>92</b>, 476–488 (1964).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaF2cXksFGru70%3D" aria-label="CAS reference 7">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 7" href="http://scholar.google.com/scholar_lookup?&title=Two%20enzymic%20mechanisms%20for%20the%20methylation%20of%20homocysteine%20by%20extracts%20of%20Escherichia%20coli&journal=Biochem.&volume=92&pages=476-488&publication_year=1964&author=Foster%2CMA&author=Tejerina%2CG&author=Guest%2CJR&author=Woods%2CDD"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="8."><p class="c-article-references__text" id="ref-CR8">Weissbach, H. & Brot, N. Regulation of methionine synthesis in <i>Escherichia coli</i>. <i>Mol. Microbiol.</i> <b>5</b>, 1593–1597 (1991).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1365-2958.1991.tb01905.x" data-track-item_id="10.1111/j.1365-2958.1991.tb01905.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1365-2958.1991.tb01905.x" aria-label="Article reference 8" data-doi="10.1111/j.1365-2958.1991.tb01905.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK3MXlsFCmt7s%3D" aria-label="CAS reference 8">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=1943695" aria-label="PubMed reference 8">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 8" href="http://scholar.google.com/scholar_lookup?&title=Regulation%20of%20methionine%20synthesis%20in%20Escherichia%20coli&journal=Mol.%20Microbiol.&doi=10.1111%2Fj.1365-2958.1991.tb01905.x&volume=5&pages=1593-1597&publication_year=1991&author=Weissbach%2CH&author=Brot%2CN"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="9."><p class="c-article-references__text" id="ref-CR9">Wolthers, K. R. <i>et al</i>. Crystal structure and solution characterization of the activation domain of human methionine synthase. <i>FEBS J.</i> <b>274</b>, 738–750 (2007).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1742-4658.2006.05618.x" data-track-item_id="10.1111/j.1742-4658.2006.05618.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1742-4658.2006.05618.x" aria-label="Article reference 9" data-doi="10.1111/j.1742-4658.2006.05618.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2sXitVOhtb8%3D" aria-label="CAS reference 9">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=17288554" aria-label="PubMed reference 9">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 9" href="http://scholar.google.com/scholar_lookup?&title=Crystal%20structure%20and%20solution%20characterization%20of%20the%20activation%20domain%20of%20human%20methionine%20synthase&journal=FEBS%20J.&doi=10.1111%2Fj.1742-4658.2006.05618.x&volume=274&pages=738-750&publication_year=2007&author=Wolthers%2CKR"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="10."><p class="c-article-references__text" id="ref-CR10">Szegedi, S. S., Castro, C. C., Koutmos, M. & Garrow, T. A. Betaine-homocysteine-<i>S</i>-methyltransferase-2 is an <i>S</i>-methylmethionine-homocysteine methyltransferase. <i>J. Biol. Chem.</i> <b>283</b>, 8939–8945 (2008).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1074/jbc.M710449200" data-track-item_id="10.1074/jbc.M710449200" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1074%2Fjbc.M710449200" aria-label="Article reference 10" data-doi="10.1074/jbc.M710449200">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1cXjslyrtLk%3D" aria-label="CAS reference 10">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=18230605" aria-label="PubMed reference 10">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276374" aria-label="PubMed Central reference 10">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 10" href="http://scholar.google.com/scholar_lookup?&title=Betaine-homocysteine-S-methyltransferase-2%20is%20an%20S-methylmethionine-homocysteine%20methyltransferase&journal=J.%20Biol.%20Chem.&doi=10.1074%2Fjbc.M710449200&volume=283&pages=8939-8945&publication_year=2008&author=Szegedi%2CSS&author=Castro%2CCC&author=Koutmos%2CM&author=Garrow%2CTA"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="11."><p class="c-article-references__text" id="ref-CR11">Kacprzak, M. M., Lewandowska, I., Matthews, R. G. & Paszewski, A. Transcriptional regulation of methionine synthase by homocysteine and choline in <i>Aspergillus nidulans</i>. <i>Biochem. J.</i> <b>376</b>, 517–524 (2003).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1042/bj20030747" data-track-item_id="10.1042/bj20030747" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1042%2Fbj20030747" aria-label="Article reference 11" data-doi="10.1042/bj20030747">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD3sXptFOqs7o%3D" aria-label="CAS reference 11">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12954077" aria-label="PubMed reference 11">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223784" aria-label="PubMed Central reference 11">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 11" href="http://scholar.google.com/scholar_lookup?&title=Transcriptional%20regulation%20of%20methionine%20synthase%20by%20homocysteine%20and%20choline%20in%20Aspergillus%20nidulans&journal=Biochem.%20J.&doi=10.1042%2Fbj20030747&volume=376&pages=517-524&publication_year=2003&author=Kacprzak%2CMM&author=Lewandowska%2CI&author=Matthews%2CRG&author=Paszewski%2CA"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="12."><p class="c-article-references__text" id="ref-CR12">Bourgis, F. <i>et al</i>. <i>S</i>-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase. <i>Plant. Cell</i> <b>11</b>, 1485–1497 (1999).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1105/tpc.11.8.1485" data-track-item_id="10.1105/tpc.11.8.1485" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1105%2Ftpc.11.8.1485" aria-label="Article reference 12" data-doi="10.1105/tpc.11.8.1485">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1MXmtVWmu7k%3D" aria-label="CAS reference 12">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10449582" aria-label="PubMed reference 12">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC144290" aria-label="PubMed Central reference 12">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 12" href="http://scholar.google.com/scholar_lookup?&title=S-methylmethionine%20plays%20a%20major%20role%20in%20phloem%20sulfur%20transport%20and%20is%20synthesized%20by%20a%20novel%20type%20of%20methyltransferase&journal=Plant.%20Cell&doi=10.1105%2Ftpc.11.8.1485&volume=11&pages=1485-1497&publication_year=1999&author=Bourgis%2CF"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="13."><p class="c-article-references__text" id="ref-CR13">Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. <i>Curr. Opin. Chem. Biol.</i> <b>1</b>, 332–339 (1997).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/S1367-5931(97)80070-1" data-track-item_id="10.1016/S1367-5931(97)80070-1" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2FS1367-5931%2897%2980070-1" aria-label="Article reference 13" data-doi="10.1016/S1367-5931(97)80070-1">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK2sXnt12mtLo%3D" aria-label="CAS reference 13">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=9667865" aria-label="PubMed reference 13">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 13" href="http://scholar.google.com/scholar_lookup?&title=Enzyme-catalyzed%20methyl%20transfers%20to%20thiols%3A%20the%20role%20of%20zinc&journal=Curr.%20Opin.%20Chem.%20Biol.&doi=10.1016%2FS1367-5931%2897%2980070-1&volume=1&pages=332-339&publication_year=1997&author=Matthews%2CRG&author=Goulding%2CCW"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="14."><p class="c-article-references__text" id="ref-CR14">Datta, S., Koutmos, M., Pattridge, K. A., Ludwig, M. L. & Matthews, R. G. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor. <i>Proc. Natl Acad. Sci. USA</i> <b>105</b>, 4115–4120 (2008).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1073/pnas.0800329105" data-track-item_id="10.1073/pnas.0800329105" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1073%2Fpnas.0800329105" aria-label="Article reference 14" data-doi="10.1073/pnas.0800329105">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2008PNAS..105.4115D" aria-label="ADS reference 14">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=18332423" aria-label="PubMed reference 14">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2393809" aria-label="PubMed Central reference 14">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 14" href="http://scholar.google.com/scholar_lookup?&title=A%20disulfide-stabilized%20conformer%20of%20methionine%20synthase%20reveals%20an%20unexpected%20role%20for%20the%20histidine%20ligand%20of%20the%20cobalamin%20cofactor&journal=Proc.%20Natl%20Acad.%20Sci.%20USA&doi=10.1073%2Fpnas.0800329105&volume=105&pages=4115-4120&publication_year=2008&author=Datta%2CS&author=Koutmos%2CM&author=Pattridge%2CKA&author=Ludwig%2CML&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="15."><p class="c-article-references__text" id="ref-CR15">Drummond, J. T., Huang, S., Blumenthal, R. M. & Matthews, R. G. Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from <i>Escherichia coli</i>. <i>Biochem.</i> <b>32</b>, 9290–9295 (1993).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/bi00087a005" data-track-item_id="10.1021/bi00087a005" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fbi00087a005" aria-label="Article reference 15" data-doi="10.1021/bi00087a005">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK3sXltlCgtr0%3D" aria-label="CAS reference 15">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 15" href="http://scholar.google.com/scholar_lookup?&title=Assignment%20of%20enzymic%20function%20to%20specific%20protein%20regions%20of%20cobalamin-dependent%20methionine%20synthase%20from%20Escherichia%20coli&journal=Biochem.&doi=10.1021%2Fbi00087a005&volume=32&pages=9290-9295&publication_year=1993&author=Drummond%2CJT&author=Huang%2CS&author=Blumenthal%2CRM&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="16."><p class="c-article-references__text" id="ref-CR16">Goulding, C. W., Postigo, D. & Matthews, R. G. Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine. <i>Biochem.</i> <b>36</b>, 8082–8091 (1997).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/bi9705164" data-track-item_id="10.1021/bi9705164" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fbi9705164" aria-label="Article reference 16" data-doi="10.1021/bi9705164">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK2sXjslahu7Y%3D" aria-label="CAS reference 16">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 16" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-dependent%20methionine%20synthase%20is%20a%20modular%20protein%20with%20distinct%20regions%20for%20binding%20homocysteine%2C%20methyltetrahydrofolate%2C%20cobalamin%2C%20and%20adenosylmethionine&journal=Biochem.&doi=10.1021%2Fbi9705164&volume=36&pages=8082-8091&publication_year=1997&author=Goulding%2CCW&author=Postigo%2CD&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="17."><p class="c-article-references__text" id="ref-CR17">Ferrer, J.-L., Ravanel, S., Robert, M. & Dumas, R. Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate. <i>J. Biol. Chem.</i> <b>279</b>, 44235–44238 (2004).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1074/jbc.C400325200" data-track-item_id="10.1074/jbc.C400325200" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1074%2Fjbc.C400325200" aria-label="Article reference 17" data-doi="10.1074/jbc.C400325200">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2cXosFymsrk%3D" aria-label="CAS reference 17">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15326182" aria-label="PubMed reference 17">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 17" href="http://scholar.google.com/scholar_lookup?&title=Crystal%20structures%20of%20cobalamin-independent%20methionine%20synthase%20complexed%20with%20zinc%2C%20homocysteine%2C%20and%20methyltetrahydrofolate&journal=J.%20Biol.%20Chem.&doi=10.1074%2Fjbc.C400325200&volume=279&pages=44235-44238&publication_year=2004&author=Ferrer%2CJ-L&author=Ravanel%2CS&author=Robert%2CM&author=Dumas%2CR"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="18."><p class="c-article-references__text" id="ref-CR18">Fu, T.-M. <i>et al</i>. Crystal structures of cobalamin-independent methionine synthase (MetE) from <i>Streptococcus mutans</i>. A dynamic zinc-inversion model. <i>J. Mol. Biol.</i> <b>412</b>, 688–697 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/j.jmb.2011.08.005" data-track-item_id="10.1016/j.jmb.2011.08.005" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2Fj.jmb.2011.08.005" aria-label="Article reference 18" data-doi="10.1016/j.jmb.2011.08.005">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXhtFyntr7E" aria-label="CAS reference 18">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21840320" aria-label="PubMed reference 18">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 18" href="http://scholar.google.com/scholar_lookup?&title=Crystal%20structures%20of%20cobalamin-independent%20methionine%20synthase%20%28MetE%29%20from%20Streptococcus%20mutans.%20A%20dynamic%20zinc-inversion%20model&journal=J.%20Mol.%20Biol.&doi=10.1016%2Fj.jmb.2011.08.005&volume=412&pages=688-697&publication_year=2011&author=Fu%2CT-M"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="19."><p class="c-article-references__text" id="ref-CR19">Pejchal, R. & Ludwig, M. L. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. <i>PLoS Biol.</i> <b>3</b>, 254–265 (2005).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1371/journal.pbio.0030254" data-track-item_id="10.1371/journal.pbio.0030254" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1371%2Fjournal.pbio.0030254" aria-label="Article reference 19" data-doi="10.1371/journal.pbio.0030254">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2MXosVeiu7w%3D" aria-label="CAS reference 19">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 19" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-independent%20methionine%20synthase%20%28MetE%29%3A%20a%20face-to-face%20double%20barrel%20that%20evolved%20by%20gene%20duplication&journal=PLoS%20Biol.&doi=10.1371%2Fjournal.pbio.0030254&volume=3&pages=254-265&publication_year=2005&author=Pejchal%2CR&author=Ludwig%2CML"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="20."><p class="c-article-references__text" id="ref-CR20">Adrian, L., Szewzyk, U., Wecke, J. & Görisch, H. Bacterial dehalorespiration with chlorinated benzenes. <i>Nat.</i> <b>408</b>, 580–583 (2000).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1038/35046063" data-track-item_id="10.1038/35046063" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1038%2F35046063" aria-label="Article reference 20" data-doi="10.1038/35046063">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2000Natur.408..580A" aria-label="ADS reference 20">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD3cXovFWrsLc%3D" aria-label="CAS reference 20">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 20" href="http://scholar.google.com/scholar_lookup?&title=Bacterial%20dehalorespiration%20with%20chlorinated%20benzenes&journal=Nat.&doi=10.1038%2F35046063&volume=408&pages=580-583&publication_year=2000&author=Adrian%2CL&author=Szewzyk%2CU&author=Wecke%2CJ&author=G%C3%B6risch%2CH"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="21."><p class="c-article-references__text" id="ref-CR21">Hölscher, T., Gorisch, H. & Adrian, L. Reductive dehalogenation of chlorobenzene congeners in cell extracts of <i>Dehalococcoides</i> sp. strain CBDB1. <i>Appl. Env. Microbiol.</i> <b>69</b>, 2999–3001 (2003).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/AEM.69.5.2999-3001.2003" data-track-item_id="10.1128/AEM.69.5.2999-3001.2003" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FAEM.69.5.2999-3001.2003" aria-label="Article reference 21" data-doi="10.1128/AEM.69.5.2999-3001.2003">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD3sXjslOltLo%3D" aria-label="CAS reference 21">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 21" href="http://scholar.google.com/scholar_lookup?&title=Reductive%20dehalogenation%20of%20chlorobenzene%20congeners%20in%20cell%20extracts%20of%20Dehalococcoides%20sp.%20strain%20CBDB1&journal=Appl.%20Env.%20Microbiol.&doi=10.1128%2FAEM.69.5.2999-3001.2003&volume=69&pages=2999-3001&publication_year=2003&author=H%C3%B6lscher%2CT&author=Gorisch%2CH&author=Adrian%2CL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="22."><p class="c-article-references__text" id="ref-CR22">Löffler, F. E. <i>et al</i>. <i>Dehalococcoides mccartyi</i> gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, <i>Dehalococcoidia</i> classis nov., order <i>Dehalococcoidales</i> ord. nov. and family <i>Dehalococcoidaceae</i> fam. nov., within the phylum <i>Chloroflexi</i>. <i>Int. J. Syst. Evol. Microbiol.</i> <b>63</b>, 625–635 (2013).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1099/ijs.0.034926-0" data-track-item_id="10.1099/ijs.0.034926-0" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1099%2Fijs.0.034926-0" aria-label="Article reference 22" data-doi="10.1099/ijs.0.034926-0">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3sXmtlWqs7Y%3D" aria-label="CAS reference 22">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=22544797" aria-label="PubMed reference 22">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 22" href="http://scholar.google.com/scholar_lookup?&title=Dehalococcoides%20mccartyi%20gen.%20nov.%2C%20sp.%20nov.%2C%20obligately%20organohalide-respiring%20anaerobic%20bacteria%20relevant%20to%20halogen%20cycling%20and%20bioremediation%2C%20belong%20to%20a%20novel%20bacterial%20class%2C%20Dehalococcoidia%20classis%20nov.%2C%20order%20Dehalococcoidales%20ord.%20nov.%20and%20family%20Dehalococcoidaceae%20fam.%20nov.%2C%20within%20the%20phylum%20Chloroflexi&journal=Int.%20J.%20Syst.%20Evol.%20Microbiol.&doi=10.1099%2Fijs.0.034926-0&volume=63&pages=625-635&publication_year=2013&author=L%C3%B6ffler%2CFE"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="23."><p class="c-article-references__text" id="ref-CR23">Zhang, Y., Rodionov, D. A., Gelfand, M. S. & Gladyshev, V. N. Comparative genomic analyses of nickel, cobalt and vitamin B<sub>12</sub> utilization. <i>BMC genomics</i> <b>10</b>, 78 (2009).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="noopener" data-track-label="10.1186/1471-2164-10-78" data-track-item_id="10.1186/1471-2164-10-78" data-track-value="article reference" data-track-action="article reference" href="https://link.springer.com/doi/10.1186/1471-2164-10-78" aria-label="Article reference 23" data-doi="10.1186/1471-2164-10-78">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1MXit1arsr8%3D" aria-label="CAS reference 23">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=19208259" aria-label="PubMed reference 23">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667541" aria-label="PubMed Central reference 23">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 23" href="http://scholar.google.com/scholar_lookup?&title=Comparative%20genomic%20analyses%20of%20nickel%2C%20cobalt%20and%20vitamin%20B12%20utilization&journal=BMC%20genomics&doi=10.1186%2F1471-2164-10-78&volume=10&publication_year=2009&author=Zhang%2CY&author=Rodionov%2CDA&author=Gelfand%2CMS&author=Gladyshev%2CVN"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="24."><p class="c-article-references__text" id="ref-CR24">Adrian, L., Rahnenführer, J., Gobom, J. & Hölscher, T. Identification of a chlorobenzene reductive dehalogenase in <i>Dehalococcoides</i> sp. strain CBDB1. <i>Appl. Env. Microbiol.</i> <b>73</b>, 7717–7724 (2007).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/AEM.01649-07" data-track-item_id="10.1128/AEM.01649-07" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FAEM.01649-07" aria-label="Article reference 24" data-doi="10.1128/AEM.01649-07">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2sXhsVeisLrP" aria-label="CAS reference 24">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 24" href="http://scholar.google.com/scholar_lookup?&title=Identification%20of%20a%20chlorobenzene%20reductive%20dehalogenase%20in%20Dehalococcoides%20sp.%20strain%20CBDB1&journal=Appl.%20Env.%20Microbiol.&doi=10.1128%2FAEM.01649-07&volume=73&pages=7717-7724&publication_year=2007&author=Adrian%2CL&author=Rahnenf%C3%BChrer%2CJ&author=Gobom%2CJ&author=H%C3%B6lscher%2CT"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="25."><p class="c-article-references__text" id="ref-CR25">Schipp, C. J., Marco-Urrea, E., Kublik, A., Seifert, J. & Adrian, L. Organic cofactors in the metabolism of <i>Dehalococcoides mccartyi</i> strains. <i>Philos. Trans. R. Soc. Lond.</i> <b>368</b>, 20120321 (2013).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1098/rstb.2012.0321" data-track-item_id="10.1098/rstb.2012.0321" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1098%2Frstb.2012.0321" aria-label="Article reference 25" data-doi="10.1098/rstb.2012.0321">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3sXlvV2gsrs%3D" aria-label="CAS reference 25">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 25" href="http://scholar.google.com/scholar_lookup?&title=Organic%20cofactors%20in%20the%20metabolism%20of%20Dehalococcoides%20mccartyi%20strains&journal=Philos.%20Trans.%20R.%20Soc.%20Lond.&doi=10.1098%2Frstb.2012.0321&volume=368&publication_year=2013&author=Schipp%2CCJ&author=Marco-Urrea%2CE&author=Kublik%2CA&author=Seifert%2CJ&author=Adrian%2CL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="26."><p class="c-article-references__text" id="ref-CR26">Kube, M. <i>et al</i>. Genome sequence of the chlorinated compound-respiring bacterium <i>Dehalococcoides</i> species strain CBDB1. <i>Nat. Biotechnol.</i> <b>23</b>, 1269–1273 (2005).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1038/nbt1131" data-track-item_id="10.1038/nbt1131" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1038%2Fnbt1131" aria-label="Article reference 26" data-doi="10.1038/nbt1131">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2MXhtVOhu7jK" aria-label="CAS reference 26">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=16116419" aria-label="PubMed reference 26">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 26" href="http://scholar.google.com/scholar_lookup?&title=Genome%20sequence%20of%20the%20chlorinated%20compound-respiring%20bacterium%20Dehalococcoides%20species%20strain%20CBDB1&journal=Nat.%20Biotechnol.&doi=10.1038%2Fnbt1131&volume=23&pages=1269-1273&publication_year=2005&author=Kube%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="27."><p class="c-article-references__text" id="ref-CR27">Seshadri, R. <i>et al</i>. Genome sequence of the PCE-dechlorinating bacterium <i>Dehalococcoides ethenogenes</i>. <i>Sci.</i> <b>307</b>, 105–108 (2005).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1126/science.1102226" data-track-item_id="10.1126/science.1102226" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1126%2Fscience.1102226" aria-label="Article reference 27" data-doi="10.1126/science.1102226">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2005Sci...307..105S" aria-label="ADS reference 27">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2MXnvFWj" aria-label="CAS reference 27">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 27" href="http://scholar.google.com/scholar_lookup?&title=Genome%20sequence%20of%20the%20PCE-dechlorinating%20bacterium%20Dehalococcoides%20ethenogenes&journal=Sci.&doi=10.1126%2Fscience.1102226&volume=307&pages=105-108&publication_year=2005&author=Seshadri%2CR"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="28."><p class="c-article-references__text" id="ref-CR28">Yi, S. <i>et al</i>. Versatility in corrinoid salvaging and remodeling pathways supports corrinoid-dependent metabolism in <i>Dehalococcoides mccartyi</i>. <i>Appl. Env. Microbiol.</i> <b>78</b>, 7745–7752 (2012).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/AEM.02150-12" data-track-item_id="10.1128/AEM.02150-12" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FAEM.02150-12" aria-label="Article reference 28" data-doi="10.1128/AEM.02150-12">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC38XhsFyis7jM" aria-label="CAS reference 28">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 28" href="http://scholar.google.com/scholar_lookup?&title=Versatility%20in%20corrinoid%20salvaging%20and%20remodeling%20pathways%20supports%20corrinoid-dependent%20metabolism%20in%20Dehalococcoides%20mccartyi&journal=Appl.%20Env.%20Microbiol.&doi=10.1128%2FAEM.02150-12&volume=78&pages=7745-7752&publication_year=2012&author=Yi%2CS"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="29."><p class="c-article-references__text" id="ref-CR29">Yan, J., Ritalahti, K. M., Wagner, D. D. & Löffler, F. E. Unexpected specificity of interspecies cobamide transfer from <i>Geobacter</i> spp. to organohalide-respiring <i>Dehalococcoides mccartyi</i> strains. <i>Appl. Env. Microbiol.</i> <b>78</b>, 6630–6636 (2012).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/AEM.01535-12" data-track-item_id="10.1128/AEM.01535-12" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FAEM.01535-12" aria-label="Article reference 29" data-doi="10.1128/AEM.01535-12">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC38XhtlalurrL" aria-label="CAS reference 29">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 29" href="http://scholar.google.com/scholar_lookup?&title=Unexpected%20specificity%20of%20interspecies%20cobamide%20transfer%20from%20Geobacter%20spp.%20to%20organohalide-respiring%20Dehalococcoides%20mccartyi%20strains&journal=Appl.%20Env.%20Microbiol.&doi=10.1128%2FAEM.01535-12&volume=78&pages=6630-6636&publication_year=2012&author=Yan%2CJ&author=Ritalahti%2CKM&author=Wagner%2CDD&author=L%C3%B6ffler%2CFE"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="30."><p class="c-article-references__text" id="ref-CR30">Tang, Y. J. <i>et al</i>. Investigation of carbon metabolism in “<i>Dehalococcoides ethenogenes</i>” strain 195 by use of isotopomer and transcriptomic analyses. <i>J. Bacteriol.</i> <b>191</b>, 5224–5231 (2009).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/JB.00085-09" data-track-item_id="10.1128/JB.00085-09" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FJB.00085-09" aria-label="Article reference 30" data-doi="10.1128/JB.00085-09">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1MXhtVSis7zP" aria-label="CAS reference 30">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=19525347" aria-label="PubMed reference 30">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725585" aria-label="PubMed Central reference 30">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 30" href="http://scholar.google.com/scholar_lookup?&title=Investigation%20of%20carbon%20metabolism%20in%20%E2%80%9CDehalococcoides%20ethenogenes%E2%80%9D%20strain%20195%20by%20use%20of%20isotopomer%20and%20transcriptomic%20analyses&journal=J.%20Bacteriol.&doi=10.1128%2FJB.00085-09&volume=191&pages=5224-5231&publication_year=2009&author=Tang%2CYJ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="31."><p class="c-article-references__text" id="ref-CR31">Marco-Urrea, E., Seifert, J., Bergen, Mvon & Adrian, L. Stable isotope peptide mass spectrometry to decipher amino acid metabolism in <i>Dehalococcoides</i> strain CBDB1. <i>J. Bacteriol.</i> <b>194</b>, 4169–4177 (2012).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1128/JB.00049-12" data-track-item_id="10.1128/JB.00049-12" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1128%2FJB.00049-12" aria-label="Article reference 31" data-doi="10.1128/JB.00049-12">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC38XhtF2itr7P" aria-label="CAS reference 31">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=22661690" aria-label="PubMed reference 31">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416212" aria-label="PubMed Central reference 31">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 31" href="http://scholar.google.com/scholar_lookup?&title=Stable%20isotope%20peptide%20mass%20spectrometry%20to%20decipher%20amino%20acid%20metabolism%20in%20Dehalococcoides%20strain%20CBDB1&journal=J.%20Bacteriol.&doi=10.1128%2FJB.00049-12&volume=194&pages=4169-4177&publication_year=2012&author=Marco-Urrea%2CE&author=Seifert%2CJ&author=Bergen%2CMvon&author=Adrian%2CL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="32."><p class="c-article-references__text" id="ref-CR32">Zhuang, W.-Q. <i>et al</i>. Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring <i>Dehalococcoides mccartyi</i>. <i>Proc. Natl Acad. Sci. USA</i> <b>111</b>, 6419–6424 (2014).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1073/pnas.1321542111" data-track-item_id="10.1073/pnas.1321542111" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1073%2Fpnas.1321542111" aria-label="Article reference 32" data-doi="10.1073/pnas.1321542111">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2014PNAS..111.6419Z" aria-label="ADS reference 32">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC2cXmtlWjtLk%3D" aria-label="CAS reference 32">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=24733917" aria-label="PubMed reference 32">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035967" aria-label="PubMed Central reference 32">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 32" href="http://scholar.google.com/scholar_lookup?&title=Incomplete%20Wood-Ljungdahl%20pathway%20facilitates%20one-carbon%20metabolism%20in%20organohalide-respiring%20Dehalococcoides%20mccartyi&journal=Proc.%20Natl%20Acad.%20Sci.%20USA&doi=10.1073%2Fpnas.1321542111&volume=111&pages=6419-6424&publication_year=2014&author=Zhuang%2CW-Q"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="33."><p class="c-article-references__text" id="ref-CR33">Schröder, I. & Thauer, R. K. Methylcobalamin:homocysteine methyltransferase from <i>Methanobacterium thermoautotrophicum</i>. <i>Eur. J. Biochem.</i> <b>263</b>, 789–796 (1999).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1046/j.1432-1327.1999.00559.x" data-track-item_id="10.1046/j.1432-1327.1999.00559.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1046%2Fj.1432-1327.1999.00559.x" aria-label="Article reference 33" data-doi="10.1046/j.1432-1327.1999.00559.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10469143" aria-label="PubMed reference 33">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 33" href="http://scholar.google.com/scholar_lookup?&title=Methylcobalamin%3Ahomocysteine%20methyltransferase%20from%20Methanobacterium%20thermoautotrophicum&journal=Eur.%20J.%20Biochem.&doi=10.1046%2Fj.1432-1327.1999.00559.x&volume=263&pages=789-796&publication_year=1999&author=Schr%C3%B6der%2CI&author=Thauer%2CRK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="34."><p class="c-article-references__text" id="ref-CR34">Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. <i>Nucleic Acids Res.</i> <b>28</b>, 27–30 (2000).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1093/nar/28.1.27" data-track-item_id="10.1093/nar/28.1.27" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1093%2Fnar%2F28.1.27" aria-label="Article reference 34" data-doi="10.1093/nar/28.1.27">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD3cXhvVGqu74%3D" aria-label="CAS reference 34">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10592173" aria-label="PubMed reference 34">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC102409" aria-label="PubMed Central reference 34">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 34" href="http://scholar.google.com/scholar_lookup?&title=KEGG%3A%20kyoto%20encyclopedia%20of%20genes%20and%20genomes&journal=Nucleic%20Acids%20Res.&doi=10.1093%2Fnar%2F28.1.27&volume=28&pages=27-30&publication_year=2000&author=Kanehisa%2CM&author=Goto%2CS"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="35."><p class="c-article-references__text" id="ref-CR35">Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. & Tanabe, M. KEGG as a reference resource for gene and protein annotation. <i>Nucleic Acids Res.</i> <b>44</b>, 457–462 (2016).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1093/nar/gkv1070" data-track-item_id="10.1093/nar/gkv1070" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1093%2Fnar%2Fgkv1070" aria-label="Article reference 35" data-doi="10.1093/nar/gkv1070">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC2sXhtV2nsrrI" aria-label="CAS reference 35">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 35" href="http://scholar.google.com/scholar_lookup?&title=KEGG%20as%20a%20reference%20resource%20for%20gene%20and%20protein%20annotation&journal=Nucleic%20Acids%20Res.&doi=10.1093%2Fnar%2Fgkv1070&volume=44&pages=457-462&publication_year=2016&author=Kanehisa%2CM&author=Sato%2CY&author=Kawashima%2CM&author=Furumichi%2CM&author=Tanabe%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="36."><p class="c-article-references__text" id="ref-CR36">Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, diseases and drugs. <i>Nucleic Acids Res.</i> <b>45</b>, 353–361 (2017).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1093/nar/gkw1092" data-track-item_id="10.1093/nar/gkw1092" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1093%2Fnar%2Fgkw1092" aria-label="Article reference 36" data-doi="10.1093/nar/gkw1092">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC1cXhslWgsrw%3D" aria-label="CAS reference 36">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 36" href="http://scholar.google.com/scholar_lookup?&title=KEGG%3A%20new%20perspectives%20on%20genomes%2C%20pathways%2C%20diseases%20and%20drugs&journal=Nucleic%20Acids%20Res.&doi=10.1093%2Fnar%2Fgkw1092&volume=45&pages=353-361&publication_year=2017&author=Kanehisa%2CM&author=Furumichi%2CM&author=Tanabe%2CM&author=Sato%2CY&author=Morishima%2CK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="37."><p class="c-article-references__text" id="ref-CR37">Wheatley, R. W., Ng, K. K. S. & Kapoor, M. Fungal cobalamin-independent methionine synthase: Insights from the model organism, <i>Neurospora crassa</i>. <i>Arch. Biochem. Biophys.</i> <b>590</b>, 125–137 (2016).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/j.abb.2015.11.037" data-track-item_id="10.1016/j.abb.2015.11.037" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2Fj.abb.2015.11.037" aria-label="Article reference 37" data-doi="10.1016/j.abb.2015.11.037">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC2MXitVansb3K" aria-label="CAS reference 37">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=26657067" aria-label="PubMed reference 37">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 37" href="http://scholar.google.com/scholar_lookup?&title=Fungal%20cobalamin-independent%20methionine%20synthase%3A%20Insights%20from%20the%20model%20organism%2C%20Neurospora%20crassa&journal=Arch.%20Biochem.%20Biophys.&doi=10.1016%2Fj.abb.2015.11.037&volume=590&pages=125-137&publication_year=2016&author=Wheatley%2CRW&author=Ng%2CKKS&author=Kapoor%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="38."><p class="c-article-references__text" id="ref-CR38">Bandarian, V. & Matthews, R. G. Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. <i>Methods Enzymol.</i> <b>380</b>, 152–169 (2004).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/S0076-6879(04)80007-7" data-track-item_id="10.1016/S0076-6879(04)80007-7" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2FS0076-6879%2804%2980007-7" aria-label="Article reference 38" data-doi="10.1016/S0076-6879(04)80007-7">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1cXhs1Omtb4%3D" aria-label="CAS reference 38">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15051336" aria-label="PubMed reference 38">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 38" href="http://scholar.google.com/scholar_lookup?&title=Measurement%20of%20energetics%20of%20conformational%20change%20in%20cobalamin-dependent%20methionine%20synthase&journal=Methods%20Enzymol.&doi=10.1016%2FS0076-6879%2804%2980007-7&volume=380&pages=152-169&publication_year=2004&author=Bandarian%2CV&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="39."><p class="c-article-references__text" id="ref-CR39">Steindal, A. H., Juzeniene, A., Johnsson, A. & Moan, J. Photodegradation of 5-methyltetrahydrofolate: biophysical aspects. <i>Photochem. Photobiol.</i> <b>82</b>, 1651–1655 (2006).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1751-1097.2006.tb09826.x" data-track-item_id="10.1111/j.1751-1097.2006.tb09826.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1751-1097.2006.tb09826.x" aria-label="Article reference 39" data-doi="10.1111/j.1751-1097.2006.tb09826.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2sXivFWqsw%3D%3D" aria-label="CAS reference 39">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=16879038" aria-label="PubMed reference 39">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 39" href="http://scholar.google.com/scholar_lookup?&title=Photodegradation%20of%205-methyltetrahydrofolate%3A%20biophysical%20aspects&journal=Photochem.%20Photobiol.&doi=10.1111%2Fj.1751-1097.2006.tb09826.x&volume=82&pages=1651-1655&publication_year=2006&author=Steindal%2CAH&author=Juzeniene%2CA&author=Johnsson%2CA&author=Moan%2CJ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="40."><p class="c-article-references__text" id="ref-CR40">Verlinde, P. H. C. J. <i>et al</i>. Influence of reducing carbohydrates on (6<i>S</i>)-5-methyltetrahydrofolic acid degradation during thermal treatments. <i>J. Agric. Food Chem.</i> <b>58</b>, 6190–6199 (2010).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/jf9041134" data-track-item_id="10.1021/jf9041134" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fjf9041134" aria-label="Article reference 40" data-doi="10.1021/jf9041134">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3cXlt1Wrsbg%3D" aria-label="CAS reference 40">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=20423084" aria-label="PubMed reference 40">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 40" href="http://scholar.google.com/scholar_lookup?&title=Influence%20of%20reducing%20carbohydrates%20on%20%286S%29-5-methyltetrahydrofolic%20acid%20degradation%20during%20thermal%20treatments&journal=J.%20Agric.%20Food%20Chem.&doi=10.1021%2Fjf9041134&volume=58&pages=6190-6199&publication_year=2010&author=Verlinde%2CPHCJ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="41."><p class="c-article-references__text" id="ref-CR41">Liu, Y. <i>et al</i>. Thermal oxidation studies on reduced folate, L-5-methyltetrahydrofolic acid (L-5-MTHF) and strategies for stabilization using food matrices. <i>J. Food Sci.</i> <b>77</b>, C236–43 (2012).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1750-3841.2011.02561.x" data-track-item_id="10.1111/j.1750-3841.2011.02561.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1750-3841.2011.02561.x" aria-label="Article reference 41" data-doi="10.1111/j.1750-3841.2011.02561.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC38XmslOjtro%3D" aria-label="CAS reference 41">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=22308962" aria-label="PubMed reference 41">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 41" href="http://scholar.google.com/scholar_lookup?&title=Thermal%20oxidation%20studies%20on%20reduced%20folate%2C%20L-5-methyltetrahydrofolic%20acid%20%28L-5-MTHF%29%20and%20strategies%20for%20stabilization%20using%20food%20matrices&journal=J.%20Food%20Sci.&doi=10.1111%2Fj.1750-3841.2011.02561.x&volume=77&pages=C236-43&publication_year=2012&author=Liu%2CY"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="42."><p class="c-article-references__text" id="ref-CR42">Parker, E. T. <i>et al</i>. Primordial synthesis of amines and amino acids in a 1958 Miller H2S-rich spark discharge experiment. <i>Proc. Natl Acad. Sci. USA</i> <b>108</b>, 5526–5531 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1073/pnas.1019191108" data-track-item_id="10.1073/pnas.1019191108" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1073%2Fpnas.1019191108" aria-label="Article reference 42" data-doi="10.1073/pnas.1019191108">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2011PNAS..108.5526P" aria-label="ADS reference 42">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21422282" aria-label="PubMed reference 42">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078417" aria-label="PubMed Central reference 42">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 42" href="http://scholar.google.com/scholar_lookup?&title=Primordial%20synthesis%20of%20amines%20and%20amino%20acids%20in%20a%201958%20Miller%20H2S-rich%20spark%20discharge%20experiment&journal=Proc.%20Natl%20Acad.%20Sci.%20USA&doi=10.1073%2Fpnas.1019191108&volume=108&pages=5526-5531&publication_year=2011&author=Parker%2CET"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="43."><p class="c-article-references__text" id="ref-CR43">Parker, E. T. <i>et al</i>. Prebiotic synthesis of methionine and other sulfur-containing organic compounds on the primitive Earth: a contemporary reassessment based on an unpublished 1958 Stanley Miller experiment. <i>Orig. Life Evol. Biosph.</i> <b>41</b>, 201–212 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="noopener" data-track-label="10.1007/s11084-010-9228-8" data-track-item_id="10.1007/s11084-010-9228-8" data-track-value="article reference" data-track-action="article reference" href="https://link.springer.com/doi/10.1007/s11084-010-9228-8" aria-label="Article reference 43" data-doi="10.1007/s11084-010-9228-8">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=2011OLEB...41..201P" aria-label="ADS reference 43">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXmtVylsbw%3D" aria-label="CAS reference 43">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21063908" aria-label="PubMed reference 43">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 43" href="http://scholar.google.com/scholar_lookup?&title=Prebiotic%20synthesis%20of%20methionine%20and%20other%20sulfur-containing%20organic%20compounds%20on%20the%20primitive%20Earth%3A%20a%20contemporary%20reassessment%20based%20on%20an%20unpublished%201958%20Stanley%20Miller%20experiment&journal=Orig.%20Life%20Evol.%20Biosph.&doi=10.1007%2Fs11084-010-9228-8&volume=41&pages=201-212&publication_year=2011&author=Parker%2CET"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="44."><p class="c-article-references__text" id="ref-CR44">Laurino, P. & Tawfik, D. S. Spontaneous emergence of <i>S</i>-adenosylmethionine and the evolution of methylation. <i>Angew. Chem. Int. Ed.</i> <b>56</b>, 343–345 (2017).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1002/anie.201609615" data-track-item_id="10.1002/anie.201609615" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2Fanie.201609615" aria-label="Article reference 44" data-doi="10.1002/anie.201609615">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC28XhvFKjsLvO" aria-label="CAS reference 44">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 44" href="http://scholar.google.com/scholar_lookup?&title=Spontaneous%20emergence%20of%20S-adenosylmethionine%20and%20the%20evolution%20of%20methylation&journal=Angew.%20Chem.%20Int.%20Ed.&doi=10.1002%2Fanie.201609615&volume=56&pages=343-345&publication_year=2017&author=Laurino%2CP&author=Tawfik%2CDS"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="45."><p class="c-article-references__text" id="ref-CR45">Decker, K., Jungermann, K. & Thauer, R. K. Energy production in anaerobic organisms. <i>Angew. Chem. Int. Ed.</i> <b>9</b>, 138–158 (1970).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1002/anie.197001381" data-track-item_id="10.1002/anie.197001381" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2Fanie.197001381" aria-label="Article reference 45" data-doi="10.1002/anie.197001381">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaE3cXps1Grug%3D%3D" aria-label="CAS reference 45">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 45" href="http://scholar.google.com/scholar_lookup?&title=Energy%20production%20in%20anaerobic%20organisms&journal=Angew.%20Chem.%20Int.%20Ed.&doi=10.1002%2Fanie.197001381&volume=9&pages=138-158&publication_year=1970&author=Decker%2CK&author=Jungermann%2CK&author=Thauer%2CRK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="46."><p class="c-article-references__text" id="ref-CR46">Martin, W. F. & Sousa, F. L. Early microbial evolution: the age of anaerobes. <i>Cold Spring Harb. Perspect. Biol.</i> <b>8</b>, a018127 (2015).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1101/cshperspect.a018127" data-track-item_id="10.1101/cshperspect.a018127" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1101%2Fcshperspect.a018127" aria-label="Article reference 46" data-doi="10.1101/cshperspect.a018127">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC28XhsFOqu73E" aria-label="CAS reference 46">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=26684184" aria-label="PubMed reference 46">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 46" href="http://scholar.google.com/scholar_lookup?&title=Early%20microbial%20evolution%3A%20the%20age%20of%20anaerobes&journal=Cold%20Spring%20Harb.%20Perspect.%20Biol.&doi=10.1101%2Fcshperspect.a018127&volume=8&publication_year=2015&author=Martin%2CWF&author=Sousa%2CFL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="47."><p class="c-article-references__text" id="ref-CR47">Dickman, S. R. Ribonucleotide reduction and the possible role of cobalamin in evolution. <i>J. Mol. Evol.</i> <b>10</b>, 251–260 (1977).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="noopener" data-track-label="10.1007/BF01764600" data-track-item_id="10.1007/BF01764600" data-track-value="article reference" data-track-action="article reference" href="https://link.springer.com/doi/10.1007/BF01764600" aria-label="Article reference 47" data-doi="10.1007/BF01764600">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=1977JMolE..10..251D" aria-label="ADS reference 47">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaE1cXntVKjsg%3D%3D" aria-label="CAS reference 47">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=599575" aria-label="PubMed reference 47">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 47" href="http://scholar.google.com/scholar_lookup?&title=Ribonucleotide%20reduction%20and%20the%20possible%20role%20of%20cobalamin%20in%20evolution&journal=J.%20Mol.%20Evol.&doi=10.1007%2FBF01764600&volume=10&pages=251-260&publication_year=1977&author=Dickman%2CSR"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="48."><p class="c-article-references__text" id="ref-CR48">Menon, S. & Ragsdale, S. W. The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein. <i>J. Biol. Chem.</i> <b>274</b>, 11513–11518 (1999).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1074/jbc.274.17.11513" data-track-item_id="10.1074/jbc.274.17.11513" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1074%2Fjbc.274.17.11513" aria-label="Article reference 48" data-doi="10.1074/jbc.274.17.11513">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1MXislGmtbg%3D" aria-label="CAS reference 48">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10206956" aria-label="PubMed reference 48">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 48" href="http://scholar.google.com/scholar_lookup?&title=The%20role%20of%20an%20iron-sulfur%20cluster%20in%20an%20enzymatic%20methylation%20reaction.%20Methylation%20of%20CO%20dehydrogenase%2Facetyl-CoA%20synthase%20by%20the%20methylated%20corrinoid%20iron-sulfur%20protein&journal=J.%20Biol.%20Chem.&doi=10.1074%2Fjbc.274.17.11513&volume=274&pages=11513-11518&publication_year=1999&author=Menon%2CS&author=Ragsdale%2CSW"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="49."><p class="c-article-references__text" id="ref-CR49">Thauer, R. K. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. <i>Microbiol.</i> <b>144</b>, 2377–2406 (1998).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1099/00221287-144-9-2377" data-track-item_id="10.1099/00221287-144-9-2377" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1099%2F00221287-144-9-2377" aria-label="Article reference 49" data-doi="10.1099/00221287-144-9-2377">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1cXmt1ygurc%3D" aria-label="CAS reference 49">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 49" href="http://scholar.google.com/scholar_lookup?&title=Biochemistry%20of%20methanogenesis%3A%20a%20tribute%20to%20Marjory%20Stephenson&journal=Microbiol.&doi=10.1099%2F00221287-144-9-2377&volume=144&pages=2377-2406&publication_year=1998&author=Thauer%2CRK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="50."><p class="c-article-references__text" id="ref-CR50">González, J. C., Peariso, K., Penner-Hahn, J. E. & Matthews, R. G. Cobalamin-independent methionine synthase from <i>Escherichia coli</i>: a zinc metalloenzyme. <i>Biochem.</i> <b>35</b>, 12228–12234 (1996).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/bi9615452" data-track-item_id="10.1021/bi9615452" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fbi9615452" aria-label="Article reference 50" data-doi="10.1021/bi9615452">Article</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 50" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-independent%20methionine%20synthase%20from%20Escherichia%20coli%3A%20a%20zinc%20metalloenzyme&journal=Biochem.&doi=10.1021%2Fbi9615452&volume=35&pages=12228-12234&publication_year=1996&author=Gonz%C3%A1lez%2CJC&author=Peariso%2CK&author=Penner-Hahn%2CJE&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="51."><p class="c-article-references__text" id="ref-CR51">Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H. & Matthews, R. G. Cobalamin-dependent methionine synthase from <i>Escherichia coli</i> B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme. <i>Biochem.</i> <b>27</b>, 8458–8465 (1988).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/bi00422a025" data-track-item_id="10.1021/bi00422a025" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fbi00422a025" aria-label="Article reference 51" data-doi="10.1021/bi00422a025">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaL1cXlvVKjs7o%3D" aria-label="CAS reference 51">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 51" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-dependent%20methionine%20synthase%20from%20Escherichia%20coli%20B%3A%20electron%20paramagnetic%20resonance%20spectra%20of%20the%20inactive%20form%20and%20the%20active%20methylated%20form%20of%20the%20enzyme&journal=Biochem.&doi=10.1021%2Fbi00422a025&volume=27&pages=8458-8465&publication_year=1988&author=Frasca%2CV&author=Banerjee%2CRV&author=Dunham%2CWR&author=Sands%2CRH&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="52."><p class="c-article-references__text" id="ref-CR52">Matthews, R. G. <i>et al</i>. Cobalamin-dependent and cobalamin-independent methionine synthases: are there two solutions to the same chemical problem? <i>Helvetica Chim. Acta</i> <b>86</b>, 3939–3954 (2003).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1002/hlca.200390329" data-track-item_id="10.1002/hlca.200390329" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2Fhlca.200390329" aria-label="Article reference 52" data-doi="10.1002/hlca.200390329">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2cXmvFCnsg%3D%3D" aria-label="CAS reference 52">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 52" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-dependent%20and%20cobalamin-independent%20methionine%20synthases%3A%20are%20there%20two%20solutions%20to%20the%20same%20chemical%20problem%3F&journal=Helvetica%20Chim.%20Acta&doi=10.1002%2Fhlca.200390329&volume=86&pages=3939-3954&publication_year=2003&author=Matthews%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="53."><p class="c-article-references__text" id="ref-CR53">Martin, B. D. & Finke, R. G. Cobalt-carbon homolysis and bond dissociation energy studies of biological alkylcobalamins: methylcobalamin, including a >1015 Co-CH3 homolysis rate enhancement at 25 °C following one-electron reduction. <i>J. Am. Chem. Soc.</i> <b>112</b>, 2419–2420 (1990).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/ja00162a053" data-track-item_id="10.1021/ja00162a053" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fja00162a053" aria-label="Article reference 53" data-doi="10.1021/ja00162a053">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK3cXhtlyrsrw%3D" aria-label="CAS reference 53">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 53" href="http://scholar.google.com/scholar_lookup?&title=Cobalt-carbon%20homolysis%20and%20bond%20dissociation%20energy%20studies%20of%20biological%20alkylcobalamins%3A%20methylcobalamin%2C%20including%20a%20%3E1015%20Co-CH3%20homolysis%20rate%20enhancement%20at%2025%E2%80%89%C2%B0C%20following%20one-electron%20reduction&journal=J.%20Am.%20Chem.%20Soc.&doi=10.1021%2Fja00162a053&volume=112&pages=2419-2420&publication_year=1990&author=Martin%2CBD&author=Finke%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="54."><p class="c-article-references__text" id="ref-CR54">Martin, B. D. & Finke, R. G. Methylcobalamin’s full- vs. half-strength cobalt-carbon σ bonds and bond dissociation enthalpies: A > 10<sup>15</sup> Co-CH<sub>3</sub> homolysis rate enhancement following one-antibonding-electron reduction of methlycobalamin. <i>J. Am. Chem. Soc.</i> <b>114</b>, 585–592 (1992).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/ja00028a027" data-track-item_id="10.1021/ja00028a027" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fja00028a027" aria-label="Article reference 54" data-doi="10.1021/ja00028a027">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK38XktF2ntQ%3D%3D" aria-label="CAS reference 54">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=20000783" aria-label="PubMed reference 54">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 54" href="http://scholar.google.com/scholar_lookup?&title=Methylcobalamin%E2%80%99s%20full-%20vs.%20half-strength%20cobalt-carbon%20%CF%83%20bonds%20and%20bond%20dissociation%20enthalpies%3A%20A%20%3E%201015%20Co-CH3%20homolysis%20rate%20enhancement%20following%20one-antibonding-electron%20reduction%20of%20methlycobalamin&journal=J.%20Am.%20Chem.%20Soc.&doi=10.1021%2Fja00028a027&volume=114&pages=585-592&publication_year=1992&author=Martin%2CBD&author=Finke%2CRG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="55."><p class="c-article-references__text" id="ref-CR55">Jensen, K. P. & Ryde, U. Conversion of homocysteine to methionine by methionine synthase. A density functional study. <i>J. Am. Chem. Soc.</i> <b>125</b>, 13970–13971 (2003).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/ja034697a" data-track-item_id="10.1021/ja034697a" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fja034697a" aria-label="Article reference 55" data-doi="10.1021/ja034697a">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD3sXotlaqsbw%3D" aria-label="CAS reference 55">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=14611228" aria-label="PubMed reference 55">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 55" href="http://scholar.google.com/scholar_lookup?&title=Conversion%20of%20homocysteine%20to%20methionine%20by%20methionine%20synthase.%20A%20density%20functional%20study&journal=J.%20Am.%20Chem.%20Soc.&doi=10.1021%2Fja034697a&volume=125&pages=13970-13971&publication_year=2003&author=Jensen%2CKP&author=Ryde%2CU"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="56."><p class="c-article-references__text" id="ref-CR56">Männel-Croisé, C. & Zelder, F. Immobilised vitamin B<sub>12</sub> as a biomimetic model for base-off/histidine-on coordination. <i>Chem. Commun.</i> <b>47</b>, 11249–11251 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1039/c1cc15093f" data-track-item_id="10.1039/c1cc15093f" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1039%2Fc1cc15093f" aria-label="Article reference 56" data-doi="10.1039/c1cc15093f">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXht1GhurvJ" aria-label="CAS reference 56">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 56" href="http://scholar.google.com/scholar_lookup?&title=Immobilised%20vitamin%20B12%20as%20a%20biomimetic%20model%20for%20base-off%2Fhistidine-on%20coordination&journal=Chem.%20Commun.&doi=10.1039%2Fc1cc15093f&volume=47&pages=11249-11251&publication_year=2011&author=M%C3%A4nnel-Crois%C3%A9%2CC&author=Zelder%2CF"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="57."><p class="c-article-references__text" id="ref-CR57">Ragsdale, S. W., Lindahl, P. A. & Münck, E. Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl-CoA by <i>Clostridium thermoaceticum</i>. <i>J. Biol. Chem.</i> <b>262</b>, 14289–14297 (1987).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaL1cXitlOm" aria-label="CAS reference 57">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=2821001" aria-label="PubMed reference 57">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 57" href="http://scholar.google.com/scholar_lookup?&title=M%C3%B6ssbauer%2C%20EPR%2C%20and%20optical%20studies%20of%20the%20corrinoid%2Firon-sulfur%20protein%20involved%20in%20the%20synthesis%20of%20acetyl-CoA%20by%20Clostridium%20thermoaceticum&journal=J.%20Biol.%20Chem.&volume=262&pages=14289-14297&publication_year=1987&author=Ragsdale%2CSW&author=Lindahl%2CPA&author=M%C3%BCnck%2CE"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="58."><p class="c-article-references__text" id="ref-CR58">Drennan, C., Huang, S., Drummond, J., Matthews, R. & Lidwig, M. How a protein binds B<sub>12</sub>. A 3.0 A X-ray structure of B<sub>12</sub>-binding domains of methionine synthase. <i>Science</i> <b>266</b>, 1669–1674 (1994).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1126/science.7992050" data-track-item_id="10.1126/science.7992050" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1126%2Fscience.7992050" aria-label="Article reference 58" data-doi="10.1126/science.7992050">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=1994Sci...266.1669L" aria-label="ADS reference 58">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK2MXis12rsb8%3D" aria-label="CAS reference 58">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=7992050" aria-label="PubMed reference 58">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 58" href="http://scholar.google.com/scholar_lookup?&title=How%20a%20protein%20binds%20B12.%20A%203.0%20A%20X-ray%20structure%20of%20B12-binding%20domains%20of%20methionine%20synthase&journal=Science&doi=10.1126%2Fscience.7992050&volume=266&pages=1669-1674&publication_year=1994&author=Drennan%2CC&author=Huang%2CS&author=Drummond%2CJ&author=Matthews%2CR&author=Lidwig%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="59."><p class="c-article-references__text" id="ref-CR59">Yan, J. <i>et al</i>. The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring <i>Dehalococcoides mccartyi</i>. <i>ISME J.</i> <b>10</b>, 1092–1101 (2016).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1038/ismej.2015.197" data-track-item_id="10.1038/ismej.2015.197" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1038%2Fismej.2015.197" aria-label="Article reference 59" data-doi="10.1038/ismej.2015.197">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC28XmvVSjsrw%3D" aria-label="CAS reference 59">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=26555247" aria-label="PubMed reference 59">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 59" href="http://scholar.google.com/scholar_lookup?&title=The%20corrinoid%20cofactor%20of%20reductive%20dehalogenases%20affects%20dechlorination%20rates%20and%20extents%20in%20organohalide-respiring%20Dehalococcoides%20mccartyi&journal=ISME%20J.&doi=10.1038%2Fismej.2015.197&volume=10&pages=1092-1101&publication_year=2016&author=Yan%2CJ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="60."><p class="c-article-references__text" id="ref-CR60">Ljungdahl, L. G. & Wood, H. G. Total synthesis of acetate from CO<sub>2</sub> by heterotrophic bacteria. <i>Annu. Rev. microbiology</i> <b>23</b>, 515–538 (1969).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1146/annurev.mi.23.100169.002503" data-track-item_id="10.1146/annurev.mi.23.100169.002503" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1146%2Fannurev.mi.23.100169.002503" aria-label="Article reference 60" data-doi="10.1146/annurev.mi.23.100169.002503">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaE3cXhvFaltQ%3D%3D" aria-label="CAS reference 60">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 60" href="http://scholar.google.com/scholar_lookup?&title=Total%20synthesis%20of%20acetate%20from%20CO2%20by%20heterotrophic%20bacteria&journal=Annu.%20Rev.%20microbiology&doi=10.1146%2Fannurev.mi.23.100169.002503&volume=23&pages=515-538&publication_year=1969&author=Ljungdahl%2CLG&author=Wood%2CHG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="61."><p class="c-article-references__text" id="ref-CR61">Matthews, R. G., Koutmos, M. & Datta, S. Cobalamin-dependent and cobamide-dependent methyltransferases. <i>Curr. Opin. Struct. Biol.</i> <b>18</b>, 658–666 (2008).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/j.sbi.2008.11.005" data-track-item_id="10.1016/j.sbi.2008.11.005" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2Fj.sbi.2008.11.005" aria-label="Article reference 61" data-doi="10.1016/j.sbi.2008.11.005">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1cXhsVOisr7P" aria-label="CAS reference 61">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=19059104" aria-label="PubMed reference 61">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2639622" aria-label="PubMed Central reference 61">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 61" href="http://scholar.google.com/scholar_lookup?&title=Cobalamin-dependent%20and%20cobamide-dependent%20methyltransferases&journal=Curr.%20Opin.%20Struct.%20Biol.&doi=10.1016%2Fj.sbi.2008.11.005&volume=18&pages=658-666&publication_year=2008&author=Matthews%2CRG&author=Koutmos%2CM&author=Datta%2CS"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="62."><p class="c-article-references__text" id="ref-CR62">Hippler, B. & Thauer, R. K. The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH. <i>FEBS Lett</i>. <b>449,</b> 165–168 (1999).</p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="63."><p class="c-article-references__text" id="ref-CR63">Ferry, J. G. Fundamentals of methanogenic pathways that are key to the biomethanation of complex biomass. <i>Curr. Opin. Biotechnol.</i> <b>22</b>, 351–357 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1016/j.copbio.2011.04.011" data-track-item_id="10.1016/j.copbio.2011.04.011" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1016%2Fj.copbio.2011.04.011" aria-label="Article reference 63" data-doi="10.1016/j.copbio.2011.04.011">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXntFSqurk%3D" aria-label="CAS reference 63">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21555213" aria-label="PubMed reference 63">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606815" aria-label="PubMed Central reference 63">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 63" href="http://scholar.google.com/scholar_lookup?&title=Fundamentals%20of%20methanogenic%20pathways%20that%20are%20key%20to%20the%20biomethanation%20of%20complex%20biomass&journal=Curr.%20Opin.%20Biotechnol.&doi=10.1016%2Fj.copbio.2011.04.011&volume=22&pages=351-357&publication_year=2011&author=Ferry%2CJG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="64."><p class="c-article-references__text" id="ref-CR64">Länge, S. & Fuchs, G. Autotrophic synthesis of activated acetic acid from CO<sub>2</sub> in <i>Methanobacterium thermoautotrophicum</i>. <i>Eur. J. Biochem.</i> <b>163</b>, 147–154 (1987).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1432-1033.1987.tb10748.x" data-track-item_id="10.1111/j.1432-1033.1987.tb10748.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1432-1033.1987.tb10748.x" aria-label="Article reference 64" data-doi="10.1111/j.1432-1033.1987.tb10748.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=3102234" aria-label="PubMed reference 64">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 64" href="http://scholar.google.com/scholar_lookup?&title=Autotrophic%20synthesis%20of%20activated%20acetic%20acid%20from%20CO2%20in%20Methanobacterium%20thermoautotrophicum&journal=Eur.%20J.%20Biochem.&doi=10.1111%2Fj.1432-1033.1987.tb10748.x&volume=163&pages=147-154&publication_year=1987&author=L%C3%A4nge%2CS&author=Fuchs%2CG"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="65."><p class="c-article-references__text" id="ref-CR65">Zhang, Y. I-TASSER server for protein 3D structure prediction. <i>BMC Bioinfomatics</i> <b>9</b>, 1–8 (2008).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="noopener" data-track-label="10.1186/1471-2105-9-1" data-track-item_id="10.1186/1471-2105-9-1" data-track-value="article reference" data-track-action="article reference" href="https://link.springer.com/doi/10.1186/1471-2105-9-1" aria-label="Article reference 65" data-doi="10.1186/1471-2105-9-1">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD1cXisFKiu7k%3D" aria-label="CAS reference 65">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 65" href="http://scholar.google.com/scholar_lookup?&title=I-TASSER%20server%20for%20protein%203D%20structure%20prediction&journal=BMC%20Bioinfomatics&doi=10.1186%2F1471-2105-9-1&volume=9&pages=1-8&publication_year=2008&author=Zhang%2CY"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="66."><p class="c-article-references__text" id="ref-CR66">DeLano, W. L. PyMol: an open-source molecular graphics tool. <i>CCP4 Newsletter on Protein Crystallography.</i> <b>40</b>, 82–92 (2002).</p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="67."><p class="c-article-references__text" id="ref-CR67">Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. <i>Mol. Biol. Evol.</i> <b>33</b>, 1870–1874 (2016).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1093/molbev/msw054" data-track-item_id="10.1093/molbev/msw054" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1093%2Fmolbev%2Fmsw054" aria-label="Article reference 67" data-doi="10.1093/molbev/msw054">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC28XhsF2ltrzN" aria-label="CAS reference 67">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=27004904" aria-label="PubMed reference 67">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210823" aria-label="PubMed Central reference 67">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 67" href="http://scholar.google.com/scholar_lookup?&title=MEGA7%3A%20molecular%20evolutionary%20genetics%20analysis%20version%207.0%20for%20bigger%20datasets&journal=Mol.%20Biol.%20Evol.&doi=10.1093%2Fmolbev%2Fmsw054&volume=33&pages=1870-1874&publication_year=2016&author=Kumar%2CS&author=Stecher%2CG&author=Tamura%2CK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="68."><p class="c-article-references__text" id="ref-CR68">Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. <i>Nucleic Acids Res.</i> <b>32</b>, 1792–1797 (2004).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1093/nar/gkh340" data-track-item_id="10.1093/nar/gkh340" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1093%2Fnar%2Fgkh340" aria-label="Article reference 68" data-doi="10.1093/nar/gkh340">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2cXisF2ks7w%3D" aria-label="CAS reference 68">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15034147" aria-label="PubMed reference 68">PubMed</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed central reference" data-track-action="pubmed central reference" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC390337" aria-label="PubMed Central reference 68">PubMed Central</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 68" href="http://scholar.google.com/scholar_lookup?&title=MUSCLE%3A%20multiple%20sequence%20alignment%20with%20high%20accuracy%20and%20high%20throughput&journal=Nucleic%20Acids%20Res.&doi=10.1093%2Fnar%2Fgkh340&volume=32&pages=1792-1797&publication_year=2004&author=Edgar%2CRC"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="69."><p class="c-article-references__text" id="ref-CR69">Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. <i>Comput. Appl. Biosci.</i> <b>8</b>, 275–282 (1992).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK38Xlt1Okt7w%3D" aria-label="CAS reference 69">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=1633570" aria-label="PubMed reference 69">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 69" href="http://scholar.google.com/scholar_lookup?&title=The%20rapid%20generation%20of%20mutation%20data%20matrices%20from%20protein%20sequences&journal=Comput.%20Appl.%20Biosci.&volume=8&pages=275-282&publication_year=1992&author=Jones%2CDT&author=Taylor%2CWR&author=Thornton%2CJM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="70."><p class="c-article-references__text" id="ref-CR70">Zuckerkandl, E. & Pauling, L. In <i>Evolving genes and proteins</i> (Elsevier), pp. 97–166 (1965).</p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="71."><p class="c-article-references__text" id="ref-CR71">Ornstein, L. & Davis, B. J. Disc electrophoresis-I: background and theory. <i>Ann. NY. Acad. Sci.</i> <b>121</b>, 321–349 (1964).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1749-6632.1964.tb14207.x" data-track-item_id="10.1111/j.1749-6632.1964.tb14207.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1749-6632.1964.tb14207.x" aria-label="Article reference 71" data-doi="10.1111/j.1749-6632.1964.tb14207.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="ads reference" data-track-action="ads reference" href="http://adsabs.harvard.edu/cgi-bin/nph-data_query?link_type=ABSTRACT&bibcode=1964NYASA.121..321O" aria-label="ADS reference 71">ADS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaF2MXksFCltQ%3D%3D" aria-label="CAS reference 71">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=14240533" aria-label="PubMed reference 71">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 71" href="http://scholar.google.com/scholar_lookup?&title=Disc%20electrophoresis-I%3A%20background%20and%20theory&journal=Ann.%20NY.%20Acad.%20Sci.&doi=10.1111%2Fj.1749-6632.1964.tb14207.x&volume=121&pages=321-349&publication_year=1964&author=Ornstein%2CL&author=Davis%2CBJ"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="72."><p class="c-article-references__text" id="ref-CR72">Holman, J. D., Tabb, D. L. & Mallick, P. Employing ProteoWizard to convert raw mass spectrometry data. <i>Curr. Protoc. Bioinforma.</i> <b>46</b>, 13.24.1–9 (2014).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1002/0471250953.bi1324s46" data-track-item_id="10.1002/0471250953.bi1324s46" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2F0471250953.bi1324s46" aria-label="Article reference 72" data-doi="10.1002/0471250953.bi1324s46">Article</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 72" href="http://scholar.google.com/scholar_lookup?&title=Employing%20ProteoWizard%20to%20convert%20raw%20mass%20spectrometry%20data&journal=Curr.%20Protoc.%20Bioinforma.&doi=10.1002%2F0471250953.bi1324s46&volume=46&pages=13.24.1-9&publication_year=2014&author=Holman%2CJD&author=Tabb%2CDL&author=Mallick%2CP"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="73."><p class="c-article-references__text" id="ref-CR73">Vaudel, M., Barsnes, H., Berven, F. S., Sickmann, A. & Martens, L. SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. <i>Proteom.</i> <b>11</b>, 996–999 (2011).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1002/pmic.201000595" data-track-item_id="10.1002/pmic.201000595" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1002%2Fpmic.201000595" aria-label="Article reference 73" data-doi="10.1002/pmic.201000595">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC3MXitFGku74%3D" aria-label="CAS reference 73">CAS</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 73" href="http://scholar.google.com/scholar_lookup?&title=SearchGUI%3A%20an%20open-source%20graphical%20user%20interface%20for%20simultaneous%20OMSSA%20and%20X%21Tandem%20searches&journal=Proteom.&doi=10.1002%2Fpmic.201000595&volume=11&pages=996-999&publication_year=2011&author=Vaudel%2CM&author=Barsnes%2CH&author=Berven%2CFS&author=Sickmann%2CA&author=Martens%2CL"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="74."><p class="c-article-references__text" id="ref-CR74">Vaudel, M. <i>et al</i>. PeptideShaker enables reanalysis of MS-derived proteomics data sets. <i>Nat. Biotechnol.</i> <b>33</b>, 22–24 (2015).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1038/nbt.3109" data-track-item_id="10.1038/nbt.3109" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1038%2Fnbt.3109" aria-label="Article reference 74" data-doi="10.1038/nbt.3109">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BC2MXmtVyhsg%3D%3D" aria-label="CAS reference 74">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=25574629" aria-label="PubMed reference 74">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 74" href="http://scholar.google.com/scholar_lookup?&title=PeptideShaker%20enables%20reanalysis%20of%20MS-derived%20proteomics%20data%20sets&journal=Nat.%20Biotechnol.&doi=10.1038%2Fnbt.3109&volume=33&pages=22-24&publication_year=2015&author=Vaudel%2CM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="75."><p class="c-article-references__text" id="ref-CR75">Sauer, K. & Thauer, R. K. Methanol:coenzyme M methyltransferase from <i>Methanosarcina barkeri</i> - substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin. <i>Eur. J. Biochem.</i> <b>261</b>, 674–681 (1999).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1046/j.1432-1327.1999.00355.x" data-track-item_id="10.1046/j.1432-1327.1999.00355.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1046%2Fj.1432-1327.1999.00355.x" aria-label="Article reference 75" data-doi="10.1046/j.1432-1327.1999.00355.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK1MXjtFCltL4%3D" aria-label="CAS reference 75">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=10215883" aria-label="PubMed reference 75">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 75" href="http://scholar.google.com/scholar_lookup?&title=Methanol%3Acoenzyme%20M%20methyltransferase%20from%20Methanosarcina%20barkeri%20-%20substitution%20of%20the%20corrinoid%20harbouring%20subunit%20MtaC%20by%20free%20cob%28I%29alamin&journal=Eur.%20J.%20Biochem.&doi=10.1046%2Fj.1432-1327.1999.00355.x&volume=261&pages=674-681&publication_year=1999&author=Sauer%2CK&author=Thauer%2CRK"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="76."><p class="c-article-references__text" id="ref-CR76">Yeo, E. J. & Wagner, C. Purification and properties of pancreatic glycine N-methyltransferase. <i>J. Biol. Chem.</i> <b>267</b>, 24669–24674 (1992).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK38Xmt1yrtrw%3D" aria-label="CAS reference 76">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=1332963" aria-label="PubMed reference 76">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 76" href="http://scholar.google.com/scholar_lookup?&title=Purification%20and%20properties%20of%20pancreatic%20glycine%20N-methyltransferase&journal=J.%20Biol.%20Chem.&volume=267&pages=24669-24674&publication_year=1992&author=Yeo%2CEJ&author=Wagner%2CC"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="77."><p class="c-article-references__text" id="ref-CR77">Patring, J. D. M., Jastrebova, J. A., Hjortmo, S. B., Andlid, T. A. & Jägerstad, I. M. Development of a simplified method for the determination of folates in baker’s yeast by HPLC with ultraviolet and fluorescence detection. <i>J. Agric. Food Chem.</i> <b>53</b>, 2406–2411 (2005).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1021/jf048083g" data-track-item_id="10.1021/jf048083g" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1021%2Fjf048083g" aria-label="Article reference 77" data-doi="10.1021/jf048083g">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DC%2BD2MXhsl2qsrc%3D" aria-label="CAS reference 77">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15796570" aria-label="PubMed reference 77">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 77" href="http://scholar.google.com/scholar_lookup?&title=Development%20of%20a%20simplified%20method%20for%20the%20determination%20of%20folates%20in%20baker%E2%80%99s%20yeast%20by%20HPLC%20with%20ultraviolet%20and%20fluorescence%20detection&journal=J.%20Agric.%20Food%20Chem.&doi=10.1021%2Fjf048083g&volume=53&pages=2406-2411&publication_year=2005&author=Patring%2CJDM&author=Jastrebova%2CJA&author=Hjortmo%2CSB&author=Andlid%2CTA&author=J%C3%A4gerstad%2CIM"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="78."><p class="c-article-references__text" id="ref-CR78">Baba, T. <i>et al</i>. Construction of <i>Escherichia coli</i> K-12 in-frame, single-gene knockout mutants: the Keio collection. <i>Mol. Sys Biol.</i> <b>2</b>, 1–11 (2006).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 78" href="http://scholar.google.com/scholar_lookup?&title=Construction%20of%20Escherichia%20coli%20K-12%20in-frame%2C%20single-gene%20knockout%20mutants%3A%20the%20Keio%20collection&journal=Mol.%20Sys%20Biol.&volume=2&pages=1-11&publication_year=2006&author=Baba%2CT"> Google Scholar</a> </p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="79."><p class="c-article-references__text" id="ref-CR79">Harwood, C. R. & Cutting, S. M. In <i>Molecular biological methods in</i> Bacillus, edited by C. R. Harwood & S. M. Cutting (Wiley, Chichester), Vol. 1, p. 548 (1990).</p></li><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="80."><p class="c-article-references__text" id="ref-CR80">Eichel, J., González, J. C., Hotze, M., Matthews, R. G. & Schröder, J. Vitamin-B<sub>12</sub>-Independent Methionine Synthase from a Higher Plant (<i>Catharanthus Roseus</i>). <i>Eur. J. Biochem.</i> <b>230</b>, 1053–1058 (1995).</p><p class="c-article-references__links u-hide-print"><a data-track="click_references" rel="nofollow noopener" data-track-label="10.1111/j.1432-1033.1995.tb20655.x" data-track-item_id="10.1111/j.1432-1033.1995.tb20655.x" data-track-value="article reference" data-track-action="article reference" href="https://doi.org/10.1111%2Fj.1432-1033.1995.tb20655.x" aria-label="Article reference 80" data-doi="10.1111/j.1432-1033.1995.tb20655.x">Article</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="cas reference" data-track-action="cas reference" href="/articles/cas-redirect/1:CAS:528:DyaK2MXmsFKgurc%3D" aria-label="CAS reference 80">CAS</a> <a data-track="click_references" rel="nofollow noopener" data-track-label="link" data-track-item_id="link" data-track-value="pubmed reference" data-track-action="pubmed reference" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=7601135" aria-label="PubMed reference 80">PubMed</a> <a data-track="click_references" data-track-action="google scholar reference" data-track-value="google scholar reference" data-track-label="link" data-track-item_id="link" rel="nofollow noopener" aria-label="Google Scholar reference 80" href="http://scholar.google.com/scholar_lookup?&title=Vitamin-B12-Independent%20Methionine%20Synthase%20from%20a%20Higher%20Plant%20%28Catharanthus%20Roseus%29&journal=Eur.%20J.%20Biochem.&doi=10.1111%2Fj.1432-1033.1995.tb20655.x&volume=230&pages=1053-1058&publication_year=1995&author=Eichel%2CJ&author=Gonz%C3%A1lez%2CJC&author=Hotze%2CM&author=Matthews%2CRG&author=Schr%C3%B6der%2CJ"> Google Scholar</a> </p></li></ol><p class="c-article-references__download u-hide-print"><a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41598-020-58873-z?format=refman&flavour=references">Download references<svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-download-medium"></use></svg></a></p></div></div></div></section></div><section data-title="Acknowledgements"><div class="c-article-section" id="Ack1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Ack1">Acknowledgements</h2><div class="c-article-section__content" id="Ack1-content"><p>We thank Benjamin Scheer for assistance with mass spectrometric experiments. Mass spectrometry was done at the Centre for Chemical Microscopy (ProVIS) at the Helmholtz Centre for Environmental Research – UFZ, which is supported by European regional development funds (EFRE-Europe Funds Saxony) and the Helmholtz Association. We thank Dr. Jochen Müller from Helmholtz Centre for Environmental Research (Environmental Biotechnology) for providing us the Quick & Easy <i>E. coli</i> Gene Deletion Kit.</p></div></div></section><section aria-labelledby="author-information" data-title="Author information"><div class="c-article-section" id="author-information-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="author-information">Author information</h2><div class="c-article-section__content" id="author-information-content"><h3 class="c-article__sub-heading" id="affiliations">Authors and Affiliations</h3><ol class="c-article-author-affiliation__list"><li id="Aff1"><p class="c-article-author-affiliation__address">Leipzig University, Institute of Biochemistry, Brüderstraße 34, 04103, Leipzig, Germany</p><p class="c-article-author-affiliation__authors-list">Darja Deobald, Rafael Hanna & Gunhild Layer</p></li><li id="Aff2"><p class="c-article-author-affiliation__address">Helmholtz Centre for Environmental Research – UFZ, Isotope Biogeochemistry, Permoserstraße 15, 04318, Leipzig, Germany</p><p class="c-article-author-affiliation__authors-list">Darja Deobald, Shahab Shahryari & Lorenz Adrian</p></li><li id="Aff3"><p class="c-article-author-affiliation__address">Freiburg University, Institute of Pharmaceutical Sciences, Stefan-Meier-Straße 19, 79104, Freiburg im Breisgau, Germany</p><p class="c-article-author-affiliation__authors-list">Rafael Hanna & Gunhild Layer</p></li><li id="Aff4"><p class="c-article-author-affiliation__address">Technische Universität Berlin, Chair of Geobiotechnology, Ackerstraße 76, 13355, Berlin, Germany</p><p class="c-article-author-affiliation__authors-list">Lorenz Adrian</p></li></ol><div class="u-js-hide u-hide-print" data-test="author-info"><span class="c-article__sub-heading">Authors</span><ol class="c-article-authors-search u-list-reset"><li id="auth-Darja-Deobald-Aff1-Aff2"><span class="c-article-authors-search__title u-h3 js-search-name">Darja Deobald</span><div class="c-article-authors-search__list"><div class="c-article-authors-search__item c-article-authors-search__list-item--left"><a href="/search?author=Darja%20Deobald" class="c-article-button" data-track="click" data-track-action="author link - 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D.D., R.H., G.L. and S.S. designed the experiments in coordination with L.A. R.H., D.D. and S.S. conducted the lab experiments. R.H. and D.D. analyzed the data. D.D. and L.A. wrote the manuscript, G.L. revised it.</p><h3 class="c-article__sub-heading" id="corresponding-author">Corresponding author</h3><p id="corresponding-author-list">Correspondence to <a id="corresp-c1" href="mailto:lorenz.adrian@ufz.de">Lorenz Adrian</a>.</p></div></div></section><section data-title="Ethics declarations"><div class="c-article-section" id="ethics-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="ethics">Ethics declarations</h2><div class="c-article-section__content" id="ethics-content"> <h3 class="c-article__sub-heading" id="FPar1">Competing interests</h3> <p>The authors declare no competing interests.</p> </div></div></section><section data-title="Additional information"><div class="c-article-section" id="additional-information-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="additional-information">Additional information</h2><div class="c-article-section__content" id="additional-information-content"><p><b>Publisher’s note</b> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></div></div></section><section data-title="Supplementary information"><div class="c-article-section" id="Sec24-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec24">Supplementary information</h2><div class="c-article-section__content" id="Sec24-content"><div data-test="supplementary-info"><div id="figshareContainer" class="c-article-figshare-container" data-test="figshare-container"></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM1"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="supplementary information." href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-020-58873-z/MediaObjects/41598_2020_58873_MOESM1_ESM.docx" data-supp-info-image="">Supplementary information.</a></h3></div></div></div></div></section><section data-title="Rights and permissions"><div class="c-article-section" id="rightslink-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="rightslink">Rights and permissions</h2><div class="c-article-section__content" id="rightslink-content"> <p><b>Open Access</b> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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id="citeas">Cite this article</h3><p class="c-bibliographic-information__citation">Deobald, D., Hanna, R., Shahryari, S. <i>et al.</i> Identification and characterization of a bacterial core methionine synthase. <i>Sci Rep</i> <b>10</b>, 2100 (2020). https://doi.org/10.1038/s41598-020-58873-z</p><p class="c-bibliographic-information__download-citation u-hide-print"><a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41598-020-58873-z?format=refman&flavour=citation">Download citation<svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-download-medium"></use></svg></a></p><ul class="c-bibliographic-information__list" data-test="publication-history"><li 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