Structure and mechanism of membrane enzymes responsible for bacterial lipid modification and polymyxin resistance

项目摘要/摘要 抗生素耐药性是对人类健康的迅速增长的威胁，有限的 开发新的抗生素。因此，迫切需要研究为新疗法的设计提供信息。 对抗抗生素耐药性上升的选择。多粘菌素是一种阳离子抗菌肽， 通过静电作用与革兰氏阴性(GN)细菌的外膜结合，并 被认为是对抗多重耐药的GN细菌感染的最后一道防线。然而，对 由于细菌的修饰，多粘菌素的形成往往相对容易。 对先天免疫系统产生的或由其他免疫系统分泌的抗菌肽(AMP)的防御 细菌种类。细菌脂多糖的脂类锚--脂类A的修饰 内毒素)修饰GN细菌的外膜，具有不同的化学成分，是一种常见的 导致抗菌剂耐药的机制。在大肠杆菌、肠杆菌和铜绿假单胞菌中，“封顶” 在含有氨基阿拉伯糖基的A脂的磷酸盐中，L-阿拉4N是主要的修饰先导 对多粘菌素和AMP的抗性。氨基阿拉伯糖“帽”是由GN细菌通过 八种蛋白质的酶传递，统称为氨基阿拉伯糖生物合成途径。这个 在很大程度上，人们对该途径的膜酶功能的机制基础知之甚少。 由于与研究在膜和膜附近起作用的酶相关的技术挑战 利用脂类底物。作为这项研究计划的一部分，我们将使用各种实验技术， 包括冷冻电子显微镜、诱变、细菌生长分析、细菌遗传学和 微尺度热渗透(MST)，以实现以下核心目标：(1)结构确定和 三种真膜酶与底物结合特性的研究 氨基阿拉伯糖生物合成途径(聚戊烯醇磷酸糖基转移酶ArnC、脱甲酰基酶 ArND和脂糖转移酶ArnT)，以及(2)酶促作用机制的研究。 正在研究的三种膜酶中的每一种的功能、金属辅因子配位和催化。 该研究计划将利用我们在膜蛋白生物化学和 结构生物学，以及从成功解决酶的几个结构中获得的经验 不与不同的脂质底物结合。该计划的影响在于其潜力：i)提供 对多种酶功能的结构基础的详细机械洞察 氨基阿拉伯糖的生物合成与GN细菌对多粘菌素的抗性，II)加深了我们对 蛋白质-脂肪与十一碳烯基磷酸的相互作用，因为研究中的所有三种酶都利用十一碳烯基 磷酸盐作为供体或受体底物，以及iii)化合物的基于结构的药物设计 能够通过靶向氨基阿拉伯糖途径的酶来恢复对多粘菌素的敏感性。

Project Summary/Abstract Antibiotic resistance is a rapidly growing threat to human health, further exacerbated by the limited development of new antibiotics. Thus, there is a dire need for research informing the design of new therapeutic options to counter the rise of antibiotic resistance. Polymyxins are cationic antimicrobial peptides that associate with the outer membrane of Gram-negative (GN) bacteria through electrostatic interactions and are considered the last line of defense against multi-drug resistant GN bacterial infections. Yet, resistance to polymyxins develops often and with relative ease, due to modifications that bacteria have developed as defenses against antimicrobial peptides (AMPs) produced by the innate immune system or secreted by other bacterial species. Modification of Lipid A, the lipidic anchor of the bacterial lipopolysaccharide (LPS or endotoxin) decorating the outer membrane of GN bacteria, with diverse chemical moieties, is a common mechanism leading to resistance to antimicrobial agents. In E. coli, S. enterica and P. aeruginosa, “capping” of the phosphates of Lipid A with an aminoarabinose moiety (L-Ara4N) is the predominant modification leading to resistance against polymyxins and AMPs. The aminoarabinose “cap” is synthesized by GN bacteria through an enzymatic relay of eight proteins collectively called the aminoarabinose biosynthetic pathway. The mechanistic basis of function for the membrane enzymes of the pathway is poorly understood, in large part due to the technical challenges associated with studying enzymes that function at or near the membrane and utilize lipidic substrates. As part of this research program, we will use a variety of experimental techniques, including cryo-electron microscopy (cryoEM), mutagenesis, bacterial growth assays, bacterial genetics, and microscale thermophoresis (MST), to achieve the following core goals: (1) Structure determination and substrate-binding characterization for the three bona fide membrane enzymes that operate in the aminoarabinose biosynthetic pathway (the polyprenol phosphate glycosyltransferase ArnC, the deformylase ArnD and the lipid-to-lipid glycosyltransferase ArnT), and (2) Investigating the mechanistic basis of enzymatic function, metal cofactor coordination, and catalysis, in each of the three membrane enzymes under study. The research program will leverage our multidisciplinary training in membrane protein biochemistry and structural biology, and experience gained from having successfully solved several structures of the enzyme ArnT bound to different lipidic substrates. The impact of the program lies within its potential to: i) Provide detailed mechanistic insights into the structural basis of a diverse set of enzymatic functions responsible for aminoarabinose biosynthesis and polymyxin resistance in GN bacteria, ii) Advance our understanding of protein-lipid interactions with undecaprenyl phosphate, as all three enzymes under study utilize undecaprenyl phosphate as either a donor or acceptor substrate, and iii) Inform structure-based drug design of compounds capable of restoring susceptibility to polymyxins by targeting enzymes of the aminoarabinose pathway.