Insights into the catalytic mechanism of formate dehydrogenases from different microbial sources

Four formate dehydrogenases (FDHs) from Pseudomonas sp . 101, Myceliophthora thermophila , Chaetomium thermophilum , and Ogataea parapolymorpha were recombinantly produced, purified, and characterized to investigate their catalytic properties and reaction mechanisms. The enzymes were studied for their ability to oxidize formate to carbon dioxide (CO 2 ) coupled with NAD + reduction. In contrast, their CO 2 reduction activity was undetectable under the tested conditions. Oxidative reactions revealed significant differences in catalytic efficiency and substrate specificity, prompting further investigation through molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations. Structural models were derived from high‐resolution structural data available for enzymes from Pseudomonas sp . 101 ( pse FDH) and Chaetomium thermophilum ( ct FDH) and extended to all four variants. Comparative analyses of the transition states revealed distinct interaction patterns within the active sites, allowing us to discriminate between high‐ and low‐performing catalysts, in full agreement with the experimental k cat values. These findings provide a mechanistic rationale for the observed disparities in catalytic performance and offer structural insights into the determinants of FDH activity. Notably, ct FDH emerged as a potential candidate for the development of CO 2 ‐reducing reactions, with QM/MM data guiding the rational design of transition‐state stabilizing mutations.