文献类型：期刊文献

英文题名：Towards the computational design and engineering of enzyme enantioselectivity: A case study by a carbonyl reductase from Gluconobacter oxydans

作者：Deng, Jian[1];Yao, Zhiqiang[1];Chen, Kangling[1];Yuan, Y. Adam[2];Lin, Jinping[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Natl Univ Singapore, Dept Biol Sci, Singapore 117543, Singapore

年份：2016

卷号：217

起止页码：31

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20154801608066);WOS:【SCI-EXPANDED(收录号:WOS:000366757900004)】；

基金：This work was financially supported by the National Key Basic Research Development Program of China ("973" Program, No. 2012CB721003), the Natural Science Foundation of China (No. 21276084), Shanghai Natural Science Foundation (No. 15ZR1408600) and National Major Science and Technology Projects of China (No. 2012ZX09304009).

语种：英文

外文关键词：Carbonyl reductase; Enantiorecognition; Computational design; OPBE; Improved enantioselectivity

摘要：In our previous work, a NAD(H)-dependent carbonyl reductase (GoCR) was identified from Gluconobacter oxydans, which showed moderate to high enantiospecificity for the reduction of different kinds of prochiral ketones. In the present study, the crystal structure of GoCR was determined at 1.65 angstrom resolution, and a computational strategy concerning substrate-enzyme docking and all-atom molecular dynamics (MD) simulation was established to help understand the molecular basis of enantiopreference and enantiorecognition for GoCR, and to further guide the design and engineering of GoCR enantioselectivity. For the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), three binding pocket residues, Cys93, Tyr149, and Trp193 were predicted to play a critical role in determining the enantioselectivity. Through site-directed mutagenesis, single-point mutant W193A was constructed and proved to reduce OPBE to ethyl (R)-2-hydroxy-4-phenylbutyrate (R-HPBE) with a significantly improved ee of >99% compared to 43.2% for the wild type (WT). Furthermore, double mutant C93 V/Y149A was proved to even invert the enantioselectivity of GoCR to afford S-HPBE at 79.8% ee. (C) 2015 Elsevier B.V. All rights reserved.