Controlling Enantioselectivity of Halohydrin Dehalogenase for Asymmetric Synthesis of Chiral Epichlorohydrin

ABSTRACT Halohydrin dehalogenases (HHDHs) are promising biocatalysts for the asymmetric synthesis of chiral epoxides. In this study, we demonstrate bidirectional enantioselectivity switch of HHDHs for the asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3‐dichloro‐2‐propanol (1,3‐DCP). HHDH from Propionicicella superfundia ( Ps HHDH) was identified for its high activity toward 1,3‐DCP with a slight preference for ( R )‐ECH formation (13.6% ee ). Guided by the catalytic mechanism of HHDHs and substrate binding features, we designed a rational engineering strategy targeting two distinct cavities within the active pocket of Ps HHDH to achieve controllable enantioselectivity. Through site‐saturation and iterative mutagenesis, we obtained two complementary variants: N87K/R88F, producing ( R )‐ECH with 96.2% ee , and N178Y/Y242F/L243Y, producing ( S )‐ECH with 80.7% ee . Molecular dynamics simulations and quantum mechanics analyses revealed that residue substitutions in these cavities reshape the S N 2 reaction trajectory, modulate halide ion release channels, and optimize active pocket volume, and thus collectively determining enantioselectivity. This strategy was successfully applied to four additional HHDHs, demonstrating its broad applicability. Notably, the N87K/R88F variant produced 90 mM ( R )‐ECH within 80 min using whole cell catalysts, highlighting its potential for industrial applications. Our work presents a structure‐guided framework for precise control of enantioselectivity in HHDHs and provides mechanistic insight into stereoselective catalysis.