文献类型：期刊文献

英文题名：Significantly enhancing the stereoselectivity of a regioselective nitrilase for the production of (S)-3-cyano-5-methylhexanoic acid using an MM/PBSA method

作者：Chen, Zhi[1];Wang, Hualei[1];Yang, Lin[1];Jiang, Shuiqing[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2021

卷号：57

期号：7

起止页码：931

外文期刊名：CHEMICAL COMMUNICATIONS

收录：;EI(收录号：20210609876685);WOS:【SCI-EXPANDED(收录号:WOS:000612511600021)】；

基金：This work was supported by the National Natural Science Foundation of China (No. 21776084/B060804) and the Fundamental Research Funds for the Central Universities (22221818014).

语种：英文

外文关键词：Binding energy - Molecular dynamics - Free energy - Hydrogen bonds

摘要：The present investigation describes the successful molecular modification of a regio- and stereo-specific nitrilase toward rac-ISBN to (S)-CMHA, a critical intermediate in the preparation of optically pure pregabalin. Two hotspots of Trp57 and Val134 were identified based on the classical binding free energy molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation method. Mutants W57F/V134M and W57Y/V134M were successfully obtained with high enantioselectivity (E>300). Furthermore, these two mutants were efficiently capable of kinetic resolution of rac-ISBN to (S)-CMHA, with both exhibiting a high e.e. (>99.9%), as well as conversion ratios of 43.8% and 40.9%, respectively. Docking and molecular dynamics simulation analysis clarified that the underlying mechanisms were related to a DC-S switch and the formation of a hydrogen bond in the active center of nitrilase. The successful utilization of the MM/PBSA method for identifying hotspots that modulate the stereoselectivity in our study could provide guidelines for the molecular modification of nitrilases, and the mutants obtained could be potentially utilized for the industrial preparation of optically pure pregabalin.