文献类型：期刊文献

英文题名：Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp PCC6803

作者：Zhang, Lujia[1];Yin, Bo[1,2,3];Wang, Chao[2,3];Jiang, Shuiqin[1];Wang, Hualei[1];Yuan, Y. Adam[2,3,4];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;[3]Natl Univ Singapore, Ctr Bioimaging Sci, Singapore 117543, Singapore;[4]Natl Univ Singapore, Suzhou Res Inst, Suzhou 215123, Jiangsu, Peoples R China

年份：2014

卷号：188

期号：2

起止页码：93

外文期刊名：JOURNAL OF STRUCTURAL BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000344839100001)】；

基金：We would like to thank H. Robinson at Brookhaven National Laboratory (X29A, NSLS) and J. He at Shanghai Synchrotron Radiation Facility (U17, SSRF) for assistance in X-Ray data collection; S. Li at Xiamen University for analytic ultracentrifugation experiments. This project was supported by seed grant from National University of Singapore (Suzhou) Research Institute (No. R-2012-N-007), Tier 1 research fund from Singapore Ministry of Education (No. R-154-000-618-112) to Y.A.Y, The Fundamental Research Funds for the Central Universities, '973' Program (No. 2012CB721003) and National Natural Science Foundation (No. 21276084/B060804) to D.W.

语种：英文

外文关键词：Crystal structure; Nitrilase; Syechocystis sp PCC6803; Substrate specificity determination

摘要：Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys-Glu-Lys catalytic triad to hydrolyze non-peptide carbon-nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 angstrom resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates. (C) 2014 Elsevier Inc. All rights reserved.