Mechanistic Insights Into the Formaldehyde Dehydrogenase‐Catalyzed Reduction of Formate: A Quantum Chemical Study

The multienzyme cascade conversion of CO ₂ to methanol is a promising route for sustainable chemical synthesis, yet the reduction of formate to formaldehyde remains its major bottleneck. Here, we use quantum chemical calculations to elucidate the catalytic mechanism of formaldehyde dehydrogenase from Burkholderia multivorans ( Bm FaldDH). The lowest‐energy pathway initiates with formate activation via a proton‐relay network involving His52, Ser49, and the NADH ribose, followed by a concerted yet asynchronous proton‐hydride transfer to yield methanediol with a barrier of 16.0 kcal mol ⁻¹ . Interestingly, calculations show that subsequent dehydration of methanediol to formaldehyde is more favorable in solution than within the active site, with barriers of 16.2 and 24.0 kcal mol ⁻¹ , respectively. This lower barrier in solution is mediated by a water‐cluster model in which three water molecules facilitate proton relay, and two stabilize the methanediol. Furthermore, binding energy calculations indicate that methanediol has a relatively weak binding affinity for Bm FaldDH, suggesting that it readily dissociates into solution. Overall, this study demonstrates that the limited efficiency of Bm FaldDH in catalyzing formate reduction stems from its incompetence to catalyze methanediol dehydration within its active pocket. These findings provide a mechanistic insight for engineering efficient enzymes for the formate‐to‐formaldehyde conversion.