Structural and Mechanistic Characterization of MraY Catalysis and Inhibition

项目摘要 抗生素耐药性是全球关注的问题。虽然细菌耐药性越来越普遍， 在过去25年中，抗生素的开发已大大缩减。这就强调了迫切需要 开发新的抗生素。革兰氏阴性菌和革兰氏阳性菌都被细胞壁包围 由肽聚糖组成，保护细胞免受渗透压的影响。肽聚糖生物合成是一个很好的- 确定了抗生素开发的目标。MraY（磷酸-MurNAc-五肽转位酶）是一种 催化细菌细胞壁生物合成的第一个膜步骤的必需膜蛋白。MraY是 许多天然产物抗生素的靶标，使其成为抗生素开发的有希望的靶标。MraY 属于聚异戊二烯磷酸N-乙酰己糖胺1-磷酸转移酶（PNPT）的亚家族 超家族PNPT超家族包括负责细胞被膜聚合物合成的酶， 细菌和真核生物中的N-连接糖基化。尽管MraY的治疗潜力和生理 PNPT超家族的重要性，对酶及其 超家族一直难以捉摸，主要是由于缺乏详细结构信息。本提案的目的是 利用结构和分子生物学方法阐明MraY的功能机制及其被天然产物抗生素抑制的机制， 功能研究。我们选择来自风生产液菌（Aquifex aeolicus，MraYAA）的MraY用于结构研究。MraYAA是一个 考虑到其活性位点的高序列保守性， 致病菌我们最近解决了MraYAA的结构，这是PNPT成员的第一个结构 家人该结构不仅提供了机械的见解，但也提出了许多新的问题。根据 在我们对MraYAA的结构和功能研究中，我们将把我们的功能研究从 致病菌为此，我们已经成功地生产了均匀和酶活性 来自病原菌的重组MraY。这些新的结果促使我们提出以下四个目标： 1)了解MraYAA催化的结构基础; 2）了解MraYAA的结构基础 天然核苷抗生素抑制; 3）MraYAA功能的酶和基于细胞的表征; 4） 病原菌MraY的生化和功能表征。这些研究将推动我们的 对了解MraY的功能和抑制机制有重要意义，这将为研究MraY的分子机制提供一个平台。 新型抗生素的未来发展。

Project Summary Antimicrobial resistance is a global concern. While bacterial resistance is becoming more widespread, antibiotic development has shrunk significantly over the past 25 years. This emphasizes a critical need for the development of new antibiotics. Both Gram-negative and Gram-positive bacteria are surrounded by cell walls made of peptidoglycan that protect the cells against osmotic pressure. Peptidoglycan biosynthesis is a well- established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) is an essential membrane protein that catalyzes the first membrane step of bacterial cell wall biosynthesis. MraY is the target of many natural product antibiotics, making it a promising target for antibiotic development. MraY belongs to a subfamily of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily. The PNPT superfamily includes enzymes responsible for cell envelope polymer synthesis in bacteria and N-linked glycosylation in eukaryotes. Despite the therapeutic potential of MraY and physiological importance of the PNPT superfamily in general, a mechanistic understanding of the enzyme and its superfamily has been elusive largely due a lack of detailed structural information. The goal of this proposal is to elucidate the mechanisms of MraY function and its inhibition by natural product antibiotics using structural and functional studies. We have chosen MraY from Aquifex aeolicus (MraYAA) for structural studies. MraYAA is an excellent model system to study given the high sequence conservation of its active site with MraYs from pathogenic bacteria. We recently solved the structure of MraYAA, the first structure of a member of the PNPT family. The structure not only provides mechanistic insights, but also raises many new questions. On the basis of our structural and functional studies of MraYAA, we will extend our functional studies to MraYs from pathogenic bacteria. Toward this end, we have managed to produce homogeneous and enzymatically active recombinant MraY from pathogenic bacteria. These new results have led us to propose the following four aims: 1) Understanding the structural basis of MraYAA catalysis; 2) Understanding the structural basis of MraYAA inhibition by natural nucleoside antibiotics; 3) Enzymatic and cell-based characterization of MraYAA function; 4) Biochemical and functional characterization of MraY from pathogenic bacteria. These studies will advance our understanding of the mechanism of MraY function and inhibition significantly, which will provide a platform for the future development of novel antibiotics.