Investigating the native functions of [NiFe]-carbon monoxide dehydrogenases through genomic context analysis

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO ₂ , representing key targets for biocatalytic CO ₂ reduction. Despite dramatic differences in catalytic rates and O ₂ tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1,376 CODH and 1,545 hybrid cluster protein sequences. Around ∼30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E, F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests environmental interdependency. These findings establish clades A, E, F as primary biocatalyst targets while defining regulatory functions for clades C, D, providing a genomics framework for predicting CODH phenotypes.