Theoretical Analysis of Selectivity Differences in Ketoreductases toward Aldehyde and Ketone Carbonyl Groups.

alcohol dehydrogenase (ADH) and ketoreductase from . CA49 (KRED12) exhibit different chemoselectivity and stereoselectivity toward a substrate with both keto and aldehyde carbonyl groups. ADH selectively reduces the keto carbonyl group while retaining the aldehyde carbonyl group, producing optically pure -alcohols. In contrast, KRED12 selectively reduces the aldehyde group and exhibits low reactivity toward ketone carbonyls. This study investigated the structural basis for these differences and the role of specific residues in the active site. Molecular dynamics (MD) simulations and quantum chemical calculations were used to investigate the interactions between the substrate and the enzymes and the essential cause of this phenomenon. The present study has revealed that ADH and KRED12 exhibit significant differences in the structure of their respective active pockets, which is a crucial determinant of their distinct chemoselectivity toward the same substrate. Moreover, residues N89, N113, and E144 within ADH as well as Q151 and D190 within KRED12 have been identified as key contributors to substrate stabilization within the active pocket through electrostatic interactions and van der Waals forces, followed by hydride transfer utilizing the coenzyme NADPH. Furthermore, the enantioselectivity mechanism of ADH has been elucidated using quantum chemical methods. Overall, these findings not only provide fundamental insights into the underlying reasons for the observed differences in selectivity but also offer a detailed mechanistic understanding of the catalytic reaction.