Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, 75123 Uppsala Sweden.
Chembiochem. 2010 Jul 5;11(10):1422-9. doi: 10.1002/cbic.201000185.
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.
随机突变靶向于马铃薯环氧化物水解酶 StEH1 的非催化活性位点残基热点，并结合酶活性筛选，分离出显示在催化水解（2,3-环氧丙基）苯时对映体偏好发生改变的酶变体。野生型酶对 S 对映体表现出 2.5:1 的比例偏好，而显示出最激进功能变化的变体对 R 对映体表现出 15:1 的偏好。该突变体积累了四个取代，分布在四个突变热点中的两个热点上：W106L、L109Y、V141K 和 I151V。对映体选择性的根本原因是 S 对映体催化水解的催化效率降低，同时保留了 R 对映体的活性。结果表明，改变这种具有生物催化相关性的酶的立体选择性是可行的。
