Disclosing Structural Determinants Governing Stereopreferences of Ketoreductases toward Reduction of Halogenated Aryl Ketones

Abstract Ketoreductases (KREDs) are widely used for asymmetric ketone reduction, yet predictable control of stereoselectivity remains challenging due to the limited understanding of structure–selectivity relationships. Here, we integrate site-directed mutagenesis and molecular dynamics simulations to elucidate key determinants of stereoselectivity within the active site of KREDs. We demonstrate that residues adjacent to the conserved PG motif contribute to shaping the catalytic pocket, together with auxiliary residues that fine-tune its architecture. Among them, a single position near the PG motif serves as an effective control point for stereoselectivity. Comparative analysis across KRED homologues demonstrates that these conserved features act as general determinants of stereochemical outcomes, enabling predictable switching between Prelog and anti-Prelog selectivity. Guided by these insights, engineered variant M3W achieves >99% (R)-selectivity toward halogenated aryl ketones while also exhibiting excellent catalytic performance in gram-scale reactions, highlighting the scalability and synthetic utility of the engineered KREDs. These results establish a simple and generalizable framework for rational engineering of enzyme stereoselectivity.