文献类型：期刊文献

英文题名：Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A

作者：Liu, Ning[1];Xiao, Hongli[1];Zang, Yongjian[2,3];Zhou, Longji[4];Mencius, Jun[4];Yang, Zhiwei[2];Quan, Shu[4];Chen, Xi[1]

机构：[1]Northwest Univ, Coll Chem & Mat Sci, Key Lab Synthet & Nat Funct Mol, Minist Educ, Xian 710127, Peoples R China;[2]Xi An Jiao Tong Univ, Sch Phys, MOE Key Lab Nonequilibrium Synth & Modulat Condens, Xian 710049, Peoples R China;[3]Yunnan Normal Univ, Inst Phys & Elect Informat, Kunming 650504, Peoples R China;[4]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2023

卷号：24

期号：23

外文期刊名：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001116970000001)】；

基金：We thank the staff of BL17B/BL18U1 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for their assistance during data collection. We also express our gratitude to Yicheng Xu for his helpful discussion with mutant design and Chu-Young Kim for scientific discussion.

语种：英文

外文关键词：natural product; Lsd18; thermostability; FoldX; Rossetta-ddG; catalytic activity

摘要：Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability.