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 the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wild-type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes 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.
Gurell
Ann
A
Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, 75123 Uppsala Sweden.
Widersten
Mikael
M
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D006868:Hydrolysis; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011198:Solanum tuberosum / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity
D004852:Epoxy Compounds; D004851:Epoxide Hydrolases; C497708:(2,3-epoxypropyl)benzene