Structural and Mechanistic Characterization of MraY Catalysis and Inhibition

MraY 催化和抑制的结构和机制表征

• 批准号: 9156353
• 负责人: Seok-Yong Lee
• 金额: $ 34.26万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9156353
• 关键词: Active Sites; Address; Affinity; Anabolism; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Architecture; Bacteria; Bacterial Antibiotic Resistance; Bacteriophages; Binding; Biochemical; Biological Assay; Biological Models; Catalysis; Cell Wall; Cells; Complex; Crystallization; Data; Dependence; Development; Enzyme Inhibition; Enzymes; Eukaryota; Family; Future; Goals; Gram-Positive Bacteria; Health; Hexosamines; Knowledge; Link; Lipids; Mass Spectrum Analysis; Membrane; Membrane Lipids; Membrane Proteins; Metal Binding Site; Modeling; Mutagenesis; Mycobacterium tuberculosis; Natural Products; Nucleosides; Osmotic Pressure; Pathway interactions; Peptidoglycan; Physiological; Polymers; Proteins; Pseudomonas aeruginosa; Public Health; Recombinants; Specificity; Staphylococcus aureus; Structure; Testing; Therapeutic; Transferase; Tunicamycin; Work; bacterial resistance; base; cell envelope; enzyme model; glycosylation; improved; inhibitor/antagonist; inorganic phosphate; insight; member; mutant; novel; paralogous gene; pathogen; pathogenic bacteria; public health relevance; translocase

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.

项目摘要 抗菌素耐药性是全球关注的问题。虽然细菌耐药性正变得越来越普遍， 在过去的25年里，抗生素的开发大幅缩水。这强调了对 开发新的抗生素。革兰氏阴性和革兰氏阳性细菌都被细胞壁包围 由保护细胞免受渗透压影响的肽聚糖制成。肽聚糖的生物合成是一个很好的- 制定了抗生素开发的目标。Mray(磷酸-MurNAc-五肽转位酶)是一种 重要的膜蛋白，催化细菌细胞壁生物合成的第一膜步骤。Mray是 许多天然产品抗生素的靶标，使其成为抗生素开发的有前途的靶标。马雷 属于聚戊烯基磷酸N-乙酰氨基己糖1-磷酸转移酶(PNPT)的一个亚家族 超级大家庭。PNPT超家族包括负责细胞膜聚合物合成的酶 细菌与真核生物中的N-连接糖基化。尽管Mray和生理学的治疗潜力 PNPT超家族的重要性，对酶和它的作用机制的理解 超级家族一直难以捉摸，主要是因为缺乏详细的结构信息。这项提议的目标是 从结构和分子水平阐明mray的作用机制及天然产物抗生素对其的抑制作用 功能研究。我们选择了Aquifex aeolicus(MraYAA)中的mray进行结构研究。MraYAA是一个 鉴于其活性中心与MraY的高度序列保守性，极好的模型系统可供研究 病原菌。我们最近解决了MraYAA的结构，这是PNPT第一个成员的结构 一家人。这种结构不仅提供了机械性的见解，还提出了许多新的问题。在此基础上 在我们对MraYAA的结构和功能的研究中，我们将把我们的功能研究从 病原菌。为此，我们已经设法生产出均一的和具有酶活性的产品 来自病原菌的重组mray。这些新的成果使我们提出了以下四个目标： 1)了解MraYAA催化的结构基础；2)了解MraYAA的结构基础 天然核苷类抗生素的抑制作用；3)MraYAA功能的酶和细胞表征；4) 病原菌Mray的生化和功能特性研究。这些研究将推动我们的 对mray功能和抑制机制的深入了解，将为进一步研究mray的作用机制提供平台。 新型抗生素的未来发展。