Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3-Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active-site residues of potato epoxide hydrolase StEH1 combined with an enzyme-activity screen allowed isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wildtype enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional change showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.