文献类型：期刊文献

英文题名：Crystal structures of Pseudomonas putida esterase reveal the functional role of residues 187 and 287 in substrate binding and chiral recognition

作者：Dou, Shuai[1,2];Kong, Xu-Dong[1,2];Ma, Bao-Di[1];Chen, Qi[1];Zhang, Jie[1];Zhou, Jiahai[2,3];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;[3]Wuhan Univ, Key Lab Combinatorial Biosynthesis & Drug Discove, Wuhan 430071, Peoples R China

年份：2014

卷号：446

期号：4

起止页码：1145

外文期刊名：BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000335367900054)】；

基金：This work was financially supported by the National Natural Science Foundation of China (No. 21276082), Ministry of Science and Technology, P.R. China (Nos. 2011CB710800,. 2011AA02A210 and 2012AA022201), and Shanghai Commission of Science and Technology (No. 11431921600). We thank Gong Y. for the excellent work in maintaining the in-house X-ray machine.

语种：英文

外文关键词：Crystal structure; Pseudomonas putida esterase; Enzyme-substrate complex; Chiral recognition; Activity enhancement; Protein engineering

摘要：A recombinant carboxylesterase (rPPE) from Pseudomonas putida ECU1011 was previously cloned and engineered to give a potential application for resolving chiral alpha-hydroxy acids including mandelic acids and derivatives. Two variants rPPE(w187H) and rPPE(w287A) showed a similar to 100-fold increase in activity towards rac-2-acetoxy-2-(2 '-chlorophenyl) acetate (rac-AcO-CPA), but rPPE(D287A) had a significant decrease in enantioselectivity (E = 8.7) compared to rPPE(w187H),4 and the wild-type rPPE (rPPE(wr)) (E > 200). Here we report the crystal structures of rPPEwr and rPPE(w187H), both by themselves and in complex with the substrate, to elucidate the structural basis of this phenomenon. An inactive mutation of nucleophile residue S159A was introduced to obtain the structure of rPPE(S159A/W187H) complexed with (S)-AcO-CPA. The structural analysis reveals that the side chain of residue Asp287 in rPPE(WT) would have a potential steric conflict with (S)-AcO-CPA when the substrate binds at the active site of the enzyme. However, the mutation W187H could facilitate the relocation of Asp287, while D287A directly eliminates the hindrance of Asp287, both of which offer sufficient space for the binding and hydrolysis of substrate. Moreover, Asp287 generates one site of the "three-point attachment model" as a hydrogen-bond donor that determines the excellent enantioselectivity of rPPE in chiral recognition, and D287A would obviously destroy the hydrogen bond and result in the low enantioselectivity of rPPE(D287A). (C) 2014 Elsevier Inc. All rights reserved.