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Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution
Author(s) -
Sun Zhoutong,
Lonsdale Richard,
Kong XuDong,
Xu JianHe,
Zhou Jiahai,
Reetz Manfred T.
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201501809
Subject(s) - stereoselectivity , stereochemistry , saturated mutagenesis , directed evolution , amino acid , desymmetrization , chemistry , docking (animal) , epoxide hydrolase , mutant , enzyme , hydrolase , binding pocket , biochemistry , combinatorial chemistry , enantioselective synthesis , gene , medicine , nursing , microsome , catalysis
Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R , R ‐ and S , S ‐selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X‐ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.