Effect of Trimethylamine‐ N ‐Oxide (TMAO) and Sorbitol on the Catalytic Activity of Candida boidinii Formate Dehydrogenase

Abstract The mechanisms by which osmolytes increase the biocatalytic efficiency of enzymes are still not well understood. In a joint computational and experimental study, we identified different effects, depending on the type of osmolyte, on the catalytic activity of formate dehydrogenase from Candida boidinii ( Cb FDH), an industrially relevant enzyme. These effects are related to enzyme inter‐domain fluctuations, solvation, and active site dynamics as well as substrate residence times. The combination of these factors could explain the experimental behavior of the k cat of Cb FDH in different osmolyte solutions, compared to buffer. Our computational studies also allowed us to propose six routes, located at the interface between both domains of Cb FDH, for water trafficking and formate exit between the active site of Cb FDH and the solvent. The results suggest that solvent‐induced changes might affect the route's dynamics and, potentially, the catalytic activity of Cb FDH. Our comprehensive analysis of protein solvation, structure, and dynamics thus provides a molecular understanding of the role of these osmolytes on enzymatic activity.