文献类型：期刊文献

英文题名：Development of an engineered carbamoyl phosphate synthetase with released sensitivity to feedback inhibition by site-directed mutation and casting error-prone PCR

作者：Shen, Su[1];Zhang, Xing[1];Li, Zhimin[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2019

卷号：129

外文期刊名：ENZYME AND MICROBIAL TECHNOLOGY

收录：;EI(收录号：20192407037142);WOS:【SCI-EXPANDED(收录号:WOS:000483412900007)】；

基金：This study was supported by the Fundamental Research Funds for the Central Universities (22221818014).

语种：英文

外文关键词：Carbamoyl phosphate synthase; Protein engineering; Feedback inhibition; Intermolecular interaction; Molecular docking

摘要：Carbamoyl phosphate synthetase (CPS) is a key enzyme in both pyrimidine and arginine biosynthesis. However, it is inhibited strongly by uridine monophosphate (UMP), which is an intermediate of the de-novo synthesis of pyrimidine nucleoside. In this study, the native carbamoyl phosphate synthetase, from Escherichia coli, was evolved by site-directed mutation and casting error-prone PCR. Compared with the wild-type, the variant N1015 F had released sensitivity to UMP and exhibited 100% of the initial activity in the presence of UMP. Variant K1006A exhibited 0.14-fold improvement in initial activity and kept above 65% of relative activity under the saturated concentration of inhibitor. Structure analysis of variants demonstrated that the reduced sensitivity to inhibitor was largely attributed to the decreased hydrogen bonds, which could reduce the binding affinity with UMP. Also, Phe with large side chain could narrow the binding pocket and generate more steric hindrance. Based on the results in this study, N1015F was an ideal alternative catalyst for the wild-type CPS for pyrimidine biosynthesis.