Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1.
Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1.
复制标题
从 MCR-1 催化域的晶体结构深入了解质粒介导的粘菌素耐药性的机制基础
DOI：
10.1038/srep39392
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发表时间：
2017-01-06
影响因子：
4.6
通讯作者：
Spencer J
中科院分区：
文献类型：
--
作者：
Hinchliffe P;Yang QE;Portal E;Young T;Li H;Tooke CL;Carvalho MJ;Paterson NG;Brem J;Niumsup PR;Tansawai U;Lei L;Li M;Shen Z;Wang Y;Schofield CJ;Mulholland AJ;Shen J;Fey N;Walsh TR;Spencer J
多粘菌素是针对广泛耐药革兰氏阴性菌的“最后一线”抗生素。最近，mcr-1基因被确定为人和动物肠杆菌科中质粒介导的耐药机制，具有广泛的地理分布和许多对多种其他抗生素耐药的生产菌株。mcr-1编码一种膜结合酶，催化磷酸乙醇胺转移到细菌脂质A上。在这里，我们目前的晶体结构揭示MCR-1周质，催化域是一个锌金属蛋白与碱性磷酸酶/硫酸酯酶倍含有三个二硫键。一种结构捕获代表转移反应中第一中间体的磷酸化形式。与锌或磷酸乙醇胺结合或催化活性有关的残基突变可恢复重组大肠杆菌对粘菌素的敏感性。杆菌缺锌可降低产MCR-1的实验室、环境、动物和人大肠杆菌中粘菌素的MIC。杆菌相反，二硫化物异构酶DsbA的过表达增加了实验室E.杆菌簇模型的初步密度泛函理论计算表明，一个单一的锌离子可能足以支持磷酸乙醇胺转移。这些数据证明了锌和二硫键对MCR-1活性的重要性，表明锌限制条件下的测定代表了对产MCR-1大肠杆菌进行表型鉴定的途径。大肠杆菌，并确定可能的催化机制的关键特征。
The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.