文献类型：期刊文献

英文题名：Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity

作者：Cui, Dongbing[1];Zhang, Lujiang[1];Yao, Zhiqiang[1];Liu, Xu[1];Lin, Jinping[1];Yuan, Adam[2];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：2013

卷号：167

期号：4

起止页码：386

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20133616705573);WOS:【SCI-EXPANDED(收录号:WOS:000324737900005)】；

基金：This work was supported by National High Technology Research and Development Program of China (NO. 2012AA020403). National Basic Research Program of China (973) (NO.2009CB724703-2012CB721003). National Natural Science Foundation of China (No.31201296). The Fundamental Research Funds for the Central Universities.

语种：英文

外文关键词：Computational design; Altered coenzyme specificity; Molecular dynamics simulation; Site-directed mutagenesis

摘要：Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2'-phosphate of NADPH, but also could enhance the binding with 2'-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2'-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity. (c) 2013 Elsevier B.V. All rights reserved.