文献类型：期刊文献

英文题名：Switching a Newly Discovered Lactonase into an Efficient and Thermostable Phosphotriesterase by Simple Double Mutations His250Ile/Ile263Trp

作者：Luo, Xiao-Jing[1];Kong, Xu-Dong[1];Zhao, Jian[1];Chen, Qi[1];Zhou, Jiahai[2];Xu, Jian-He[1]

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, Shanghai 200032, Peoples R China

年份：2014

卷号：111

期号：10

起止页码：1920

外文期刊名：BIOTECHNOLOGY AND BIOENGINEERING

收录：;EI(收录号：20143518114240);WOS:【SCI-EXPANDED(收录号:WOS:000341236400002)】；

基金：Contract grant sponsor: Ministry of Science and TechnologyGrant numbers: 2012AA022206C; 2011CB710800Contract grant sponsor: Ministry of EducationContract grant number: 222201314016

语种：英文

外文关键词：organophosphate; phosphotriesterase; lactonase; site-directed mutagenesis; functional switch; X-ray crystallography

摘要：OPHC2 is a thermostable organophosphate (OP) hydrolase in the beta-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethylparaoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 x 10(7)-fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 angstrom resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPH(M2) from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1)) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in beta-lactamase superfamily and provide efficient and robust enzymes for OP detoxification. (C) 2014 Wiley Periodicals, Inc.