文献类型：期刊文献

英文题名：Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

作者：Kong, Xu-Dong[1,2];Yuan, Shuguang[2];Li, Lin[3];Chen, She[3];Xu, Jian-He[1];Zhou, Jiahai[2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Bioorgan & Nat Prod Chem, Shanghai 200032, Peoples R China;[3]Natl Inst Biol Sci, Beijing 102206, Peoples R China

年份：2014

卷号：111

期号：44

起止页码：15717

外文期刊名：PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000344088100036)】；

基金：We are grateful for access to beamline BL17U1 at Shanghai Synchrotron Radiation Facility and thank the beamline staff for technical support. We also thank Dr. Peter K. Park and Profs. Zhihong Guo and Ran Hong for helpful discussions. This work was supported by National Program on Key Basic Research of China Grant 2011CB710800 (to J.-H.X. and J.Z.), National Grand Project for Medicine Innovation Grant 2012ZX10002006 (to J.Z.), National Natural Science Foundation of China Grant 21276082 (to J.-H.X.), and a grant from the Open Fund from the State Key Laboratory of Bioreactor Engineering (to J.Z.).

语种：英文

外文关键词：epoxide hydrolase; X-ray crystallography; protein engineering; product release; bulky substrate

摘要：Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other beta-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward alpha-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward a-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.