Engineering the Stereoselectivity of Short-Chain Dehydrogenases/Reductases for “Difficult-to-Reduce” Ketones Reduction via Electrostatic Tuning and Pocket Confinement

Chiral alcohols derived from “difficult-to-reduce” ketones are key precursors in pharmaceutical intermediates and high-value fine chemicals. Despite advances in alcohol dehydrogenases (ADHs), the stereoselective reduction of “difficult-to-reduce” ketones remains a major challenge. In this study, we identified 10 short-chain dehydrogenases/reductases (SDRs) through gene mining using tetrahydrofuran-3-one and tetrahydrothiophene-3-one as substrates. Structure-guided mutagenesis combined with docking and molecular dynamics simulations revealed key residues regulating enantiomer selectivity. The A3-E145W mutation enhanced S-selectivity for tetrahydrofuran-3-one by optimizing electrostatic and aromatic stabilization, whereas the A3-A94N/E145S mutant achieved >99% ee (R) for tetrahydrothiophene-3-one through strengthened hydrogen bonding and steric gating. Taken together, these results reveal a general mechanism by which electrostatic tuning and pocket confinement synergistically influence SDR stereoselectivity, providing a versatile strategy for engineering biocatalysts for challenging ketone reduction reactions.