FIG 1.

(A) The Wood-Ljungdahl pathway and the conversion of its product acetyl-CoA to ethanol, acetate, and butyrate. Enzyme abbreviations are given in blue and gene names are in black with locus tags (19) in red. H₄F stands for tetrahydrofolate. Separate methyl and carbonyl branches converge in the formation of acetyl-CoA. In the methyl branch, a reversible hydrogen-dependent CO₂ reductase complex (HDCR), which contains formate dehydrogenase (fdh) and [Fe-Fe] hydrogenase components, reduces CO₂ to formate using H₂ as its direct electron donor. Formate is activated to produce N¹⁰-formyl-H₄F (HCO-H₄F) in an ATP-requiring reaction by N¹⁰-formyl-H₄F synthetase (FTS). N⁵,N¹⁰-methenyl-H₄F (CH≡H₄F) is then generated via cyclization and dehydration by N⁵,N¹⁰-methenyl-tetrahydrofolate 5-hydrolase (formyl-H₄F cyclohydrolase; fchA) (MTC). Bifunctional FolD protein, which includes N⁵,N¹⁰-methylene-H₄F dehydrogenase (MTD) and additional formyl-H₄F cyclohydrolase activity, converts CH≡H₄F to N⁵,N¹⁰-methylene-H₄F (CH₂=H₄F), which is then further reduced to N⁵-methyl-H₄F (CH₃-H₄F) by N⁵,N¹⁰-methylene-H₄F reductase (MTR) with catalytic subunit MetF and an accessory Fe/S- and Zn-containing subunit MetV. CH₃-H₄F then transfers its methyl group to a corrinoid iron-sulfur protein to form Co³⁺-methyl corrinoid iron-sulfur protein (CH₃-CFeSP) catalyzed by CH₃-H₄F:corrinoid methyltransferase (MeTr). In the carbonyl branch, carbon monoxide dehydrogenase (CODH) produces CO contained within a protein channel, depicted as [CO], by reduction of CO₂ using low-potential reduced ferredoxin (${\text{Fd}}_{\text{red}}^{-2}$). Acetyl-CoA, shown at the center in the shaded oval, is then produced by condensation of the corrinoid methyl and channel-bound CO groups by acetyl-CoA synthase (ACS) acsB. In the pathway leading to ethanol, acetyl-CoA is reduced in two steps by bifunctional acetaldehyde-CoA/alcohol dehydrogenase (AdhE). An alternative pathway would be to convert acetate to ethanol by way of aldehyde dehydrogenase (Aldh). Acetate formation from acetyl-CoA is via phosphotransacetylase (Pta/Ptb) that produces acetyl phosphate (CD2683 is annotated in the KEGG database as a putative phosphotransacetylase ortholog, and other candidates potentially active with acetate could include phosphotransbutyrylase CD0112 and the two other annotated ptb homologs CD0715 and CD2425). Acetyl phosphate is then used to generate ATP by acetate kinase or butyrate kinase active with acetate (Ack/Buk). Formation of butyrate follows the typical pathway in which thiolase (acetyl-CoA acetyltransferase) (THL) generates acetoacetyl-CoA that is acted on by 3-hydroxybutyryl-CoA dehydrogenase (HBD) followed by 3-hydroxybutyryl-CoA dehydratase (crotonase) (CRT) to form crotonyl-CoA. The electron-bifurcating butyryl-CoA dehydrogenase (Bcd) CD1054 in complex with electron-transferring flavoprotein subunits EtfB and EtfA (CD1055 and CD1056), complex (Bcd-EtfAB)₄, then generates butyryl-CoA (75–77). Thereafter, butyrate is produced by butyryl-CoA:acetate CoA-transferase activity (CoA transfer) using free acetate to reform one molecule of acetyl-CoA. (B) The Wood-Ljungdahl pathway operon in C. difficile. A 15-gene, 18.4-kb region (CD0716 to CD0730) is shown according to data in references 19 and 20. Gene designations and corresponding products are indicated.