Regioselectivity Engineering of Epoxide Hydrolase: Near-Perfect Enantioconvergence through a Single Site Mutation

An epoxide hydrolase from Vigna radiata (VrEH2) affords partial enantioconvergence (84% ee) in the enzymatic hydrolysis of racemic p-nitrostyrene oxide (pNSO), mainly due to insufficient regioselectivity for the (S)-enantiomer (r(s) = alpha(s)/beta(s) = 7.3). To improve the (S)-pNSO regioselectivity, a small but smart library of VrEH2 mutants was constructed by substituting each of four key residues lining the substrate binding site with a simplified amino acid alphabet of Val, Asn, Phe, and Trp. Among the mutants, M263N attacked almost exclusively at C alpha in the (S)-epoxide ring with satisfactory regioselectivity (r(s) = 99.0), without compromising the original high regioselectivity for the (R)-epoxide (r(R) = 99.0), resulting in near-perfect enantioconvergence (>99% analytical yield, 98% ee). Structural and conformational analysis showed that the introduced Asn263 formed additional hydrogen bonds with the nitro group in substrate, causing a shift in the substrate binding pose. This shift increased the difference in attacking distances between C alpha and C beta, leading to an improved regiopreference toward (S)-pNSO and affording near-perfect enantioconvergence.