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All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">M. thermoacetica<\/jats:named-content>gene that putatively encodes this enzyme,<jats:italic>metF<\/jats:italic>, is part of a transcription unit also containing the genes<jats:italic>hdrCBA<\/jats:italic>,<jats:italic>mvhD<\/jats:italic>, and<jats:italic>metV<\/jats:italic>. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the<jats:italic>metF<\/jats:italic>gene in<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">Escherichia coli<\/jats:named-content>revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in<jats:named-content xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" content-type=\"genus-species\" xlink:type=\"simple\">M. thermoacetica<\/jats:named-content>is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.<\/jats:p>","DOI":"10.1128\/jb.01839-14","type":"journal-article","created":{"date-parts":[[2014,7,8]],"date-time":"2014-07-08T03:49:32Z","timestamp":1404791372000},"page":"3303-3314","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":102,"title":["Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica"],"prefix":"10.1128","volume":"196","author":[{"given":"Johanna","family":"Mock","sequence":"first","affiliation":[{"name":"Max Planck Institute for Terrestrial Microbiology, Marburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuning","family":"Wang","sequence":"additional","affiliation":[{"name":"Max Planck Institute for Terrestrial Microbiology, Marburg, Germany"},{"name":"State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiyan","family":"Huang","sequence":"additional","affiliation":[{"name":"Max Planck Institute for Terrestrial Microbiology, Marburg, Germany"},{"name":"Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00f6rg","family":"Kahnt","sequence":"additional","affiliation":[{"name":"Max Planck Institute for Terrestrial Microbiology, Marburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rudolf K.","family":"Thauer","sequence":"additional","affiliation":[{"name":"Max Planck Institute for Terrestrial Microbiology, Marburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"crossref","DOI":"10.1007\/978-1-4615-1777-1","volume-title":"Acetogenesis","author":"Drake HL","year":"1994","unstructured":"DrakeHL. 1994. 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