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Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene
Author(s) -
Gurell Ann,
Widersten Mikael
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000185
Subject(s) - enantiomer , epoxide hydrolase , chemistry , stereoselectivity , stereochemistry , hydrolase , epoxide , enzyme , active site , hydrolysis , substrate (aquarium) , mutagenesis , benzene , catalysis , mutant , biochemistry , organic chemistry , biology , gene , ecology , microsome
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.