文献类型：期刊文献

英文题名：Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

作者：Sun, Zhoutong[1,2];Lonsdale, Richard[1,2];Kong, Xu-Dong[3];Xu, Jian-He[3];Zhou, Jiahai[4];Reetz, Manfred T.[1,2]

机构：[1]Max Planck Inst Kohlenforsch, D-45470 Mulheim, Germany;[2]Univ Marburg, Fachbereich Chem, D-35032 Marburg, Germany;[3]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[4]Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Bioorgan & Nat Prod Chem, Shanghai 200032, Peoples R China

年份：2015

卷号：54

期号：42

起止页码：12410

外文期刊名：ANGEWANDTE CHEMIE-INTERNATIONAL EDITION

收录：;EI(收录号：20151700776640);WOS:【SCI-EXPANDED(收录号:WOS:000363397300028)】；

基金：This research was supported by the Max Planck Society, the LOEWE Research Cluster SynChemBio, and the Arthur C. Cope Fund, in addition to grants from the National Program on Key Basic Research of China (2011CB7710800).

语种：英文

外文关键词：amino acid alphabet; directed evolution; epoxide hydrolases; saturation mutagenesis; stereoselectivity

摘要：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.