Rational engineering of ketoreductase SsSDR1 for stereoselectivity inversion toward 3-N-substituted azacyclic ketones.

The stereocomplementary reduction of 3-N-substituted azacyclic ketones using ketoreductases remains inherently challenging, especially for the simultaneous achievement of strict stereoselectivity reversal toward both five- and six-membered ring substrates. In this study, ketoreductase SsSDR1, which exhibits strict (R)-configuration stereoselectivity toward 3-N-substituted azacyclic ketones, was selected for engineering to invert its stereoselectivity. By adopting a comprehensive rational engineering strategy that combines adjustment of the size of the substrate-binding pocket and introduction of favorable intermolecular interactions, an optimized mutant SsSDR1-M4 (G94W/H145A/Y188A/L205A) was successfully screened. Stereocomplementary reduction reactions of N-Boc-piperidone (1a) and N-Boc-pyrrolidinone (1b) were performed on a 50 mL scale using SsSDR1-WT and SsSDR1-M4 as biocatalysts, respectively. The results demonstrated that SsSDR1-M4 not only catalyzes the formation of strictly (S)-2a (99.0 % ee) and (S)-2b (>99.9 % ee) but also exhibits higher catalytic activity than the wild-type enzyme. To elucidate the molecular mechanisms underlying the enhanced catalytic activity and reversed stereoselectivity of SsSDR1-M4, a combination of molecular docking, 2D interaction analysis, and molecular dynamics (MD) simulations was employed. Notably, the substrate-binding pocket of SsSDR1-M4 forms a "hydrophobic clamp", where residues W94 and Y246 at both ends of the clamp establish hydrophobic interactions with the N-heterocycle and 3-substituent of the substrate, respectively, thereby stabilizing the pro-S conformation of the substrate. This mechanism enables SsSDR1-M4 to universally reverse the stereoselectivity towards 3-N-substituted azacyclic ketones compared to the wild type. This study achieved precise regulation of stereoselectivity of ketoreductase toward 3-N-substituted azacyclic ketones, offering deeper insights into the structural and functional dynamics of ketoreductases.