Identification of Residue Involved in Nucleotidyltransferase Activity of LinA from Staphylococci by Site-directed Mutagenesis

作     者：ZOU Xiao-Ming JIANG Mei-Qin ZHAO Yan-He SUN Li-Fang YANG Wen-Di YE Xian-Ren WU Yun-Kun 邹小明;蒋梅琴;赵艳和;孙丽芳;杨闻笛;叶先仁;吴允昆

作者机构：State Key Laboratory of Structural Chemistry Fujian Institute of Research on the Structure of Matter Chinese Academy of Sciences Department of Cell Biology and Biophysics University of Texas Southwestern Medical Center Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation College of Life Science Fujian Normal University Fujian Cancer Hospital & Fujian Medical University Cancer Hospital 

出 版 物：《Chinese Journal of Structural Chemistry》 (结构化学（英文）)

年 卷 期：2019年第38卷第3期

页      面：422-428页

核心收录：

学科分类：0817[工学-化学工程与技术] 08[工学] 

基　　金：supported by the National Natural Science Foundation of China(31470741) Natural Science Foundation of Fujian Province(2016J01173) the Key Project of Fujian Province(2017N0031) the STS project of Chinese Academy of Sciences and Fujian Province(2016T3041) the medical talent training project of Fujian provincial health and family planning commission(2016-ZQN-19) National Thousand Talents Program of China 

主　　题：LinA nucleotidyltransferase site-directed mutagenesis enzymatic assay ITC 

摘      要：The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.