Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase
Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase
复制标题
DOI：
10.7554/elife.56707
复制
发表时间：
2020-06-08
期刊：
影响因子：
7.7
通讯作者：
Tokuriki, Nobuhiko
中科院分区：
文献类型：
--
作者：
Chen, John Z.;Fowler, Douglas M.;Tokuriki, Nobuhiko
金属- β -内酰胺酶（MBLs）可降解广谱的β -内酰胺类抗生素，是耐多药细菌的主要传播源。尽管对不同的MBLs进行了许多生化研究，但对残基在酶的稳定性和功能，特别是底物特异性中的作用的分子理解仍然缺乏。在这里，我们采用深度突变扫描（DMS），通过测量数千个VIM-2突变体在两种不同温度（25℃和37℃）下对三种代表性的β -内酰胺类药物（氨苄西林、头孢噻肟和美罗培南）降解的影响，生成临床主要MBL VIM-2的综合单氨基酸变异数据。我们揭示了负责表达和易位的残基，以及增加耐药性和/或改变底物特异性的突变。特异性改变突变的分布揭示了三种底物的不同分子识别。此外，在自然发生的变异中经常观察到这些改变功能的突变，这表明酶不断进化成为更有效的抗性基因。
Metallo-beta-lactamases (MBLs) degrade a broad spectrum of beta-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in diverse MBLs, molecular understanding of the roles of residues in the enzyme's stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of beta-lactams (ampicillin, cefotaxime, and meropenem) and at two different temperatures (25 degrees C and 37 degrees C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes have continuously evolved to become more potent resistance genes.