Structure and function of a metabolic pacemaker in bacterial cell membrane

细菌细胞膜代谢起搏器的结构和功能

基本信息

批准号：
10457395
负责人：
Lei Zheng
金额：
$ 31.98万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10457395
关键词：
Active Sites Anabolism Anaerobic Bacteria Antibiotic Resistance Attenuated Bacteria Bacterial Infections Binding Binding Sites Biochemical Biochemistry Biological Assay Calorimetry Carbon Catalysis Cell Survival Cell membrane Chemicals Chemistry Communicable Diseases Computer Models Cryoelectron Microscopy Crystallization Cysteine Data Development Diffusion Disease Resistance Disulfides Environment Enzymes Equilibrium Escherichia coli Eukaryota Family Fermentation Fluorescence Fluorescence Resonance Energy Transfer Gene Expression Genetic Transcription Glucose Glucose Transporter Glutathione Glutathione Disulfide Glycolysis Gram-Negative Bacteria Growth Homeostasis Homologous Protein In Situ In Vitro Integral Membrane Protein Intracellular Membranes Label Lead Lipid Bilayers Lipids Measures Mediating Membrane Metabolic Metabolic Pathway Metabolism Methods Microbiology Molecular Molecular Conformation Monitor Mutation Oxidation-Reduction Oxides Pacemakers Pathway interactions Phosphatidylglycerols Phospholipids Phosphoric Monoester Hydrolases Prokaryotic Cells Protein Conformation Protein phosphatase Proteins Regulation Reporting Resolution Roentgen Rays Role Signal Transduction Site Structure Surface Testing Titrations Virulence X-Ray Crystallography Yang aminoacid biosynthesis antimicrobial bacterial metabolism base cell growth cell type crosslink dimer family structure genetic approach glucose uptake inorganic phosphate insight metabolomics microorganism mutant nanodisk novel novel therapeutic intervention particle pathogen prevent response unnatural amino acids vapor

项目摘要

Abstract Glycolysis constitutes one of the most important metabolic pathways conserved in both eukaryotes and prokaryotes. In the pathway, glucose is broken down to form small 3-carbon phosphate metabolites essential for cell growth and survival. In microorganisms, properly maintaining glycolysis is important for the development of bacterial infection and virulence and antibiotic resistance. In this project, we aim to study the structure and function of phosphatidylglycerol phosphatase PgpA to elucidate a novel regulatory mechanism of glycolysis in bacterium. PgpA is an integral membrane protein ubiquitously found in Gram-negative bacterium. We found that PgpA functions as a moonlighting enzyme; i.e. PgpA is not only involved in phospholipid biosynthesis but also acts as an essential metabolic regulator by hydrolyzing the key 3-carbon phosphate glycolytic metabolites in E. coli. Mutational inactivation of PgpA in E. coli greatly facilitates bacterial metabolism and growth. We have also identified a novel redox-regulatory mechanism of PgpA, which is important to maintain bacterial metabolic homeostasis. Our findings raise the hypothesis for a redox-mediated regulatory mechanism in which PgpA regulates bacterial glycolysis by controlling glutathione-mediated redox balance based on external and internal metabolic signals. This regulatory mechanism is novel and has not yet been reported in any cell type. To further understand this regulatory mechanism, we will study how PgpA controls bacterial glucose uptake and regulate glycolytic activity using a combination of biochemistry, microbiology, and metabolomic approaches.To understand how PgpA regulates intracellular redox balance, we will examine glutathione biosynthesis and monitor redox changes on the membrane surface of PgpA to demonstrate how PgpA uses an integrative “Ying- Yang” mechanism to achieve both metabolic homeostasis and redox balance. We also found the redox-mediated regulation of PgpA is mediated by dimeric disulfide crosslinking within PgpA dimer. To gain structural insights into this novel redox-regulated catalytic mechanism, we will study the catalytic activity of PgpA and co-factor Mg2+ binding in response to redox changes in vitro using biochemical assays. We will also study this molecular mechanism using FRET to demonstrate how dimeric crosslinking alters protein conformation to allosterically change the active site conformation in order to control the PgpA catalysis. Since no structure is available in the PgpA family, we will determine the structures of PgpA in two distinct redox (active/inactivated) states using the X-ray crystallography and single-particle cryoEM approaches to establish a structural basis for the redox- regulated catalytic mechanism of PgpA. This mechanism is conserved in many Gram-negative pathogens. Our studies will reveal an important mechanism to understand metabolic regulation in microorganisms.

抽象的糖酵解构成构成的最重要的代谢途径之一，在真核生物和原核生物。在途径中，葡萄糖被分解以形成小的3碳磷酸代谢产物细胞生长和生存。在微生物中，适当维持糖酵解对于发展很重要细菌感染和毒力和抗生素耐药性。在这个项目中，我们旨在研究结构和磷脂酰甘油磷酸酶PGPA的功能阐明了糖酵解中新型调节机制细菌。 PGPA是一种在革兰氏阴性细菌中普遍存在的整体膜蛋白。我们发现 PGPA充当月光酶；即PGPA不仅参与磷脂生物合成，而且还参与通过水解E. 大肠杆菌。大肠杆菌中PGPA的突变灭活极为最喜欢的细菌代谢和生长。我们也有确定了一种新型PGPA的氧化还原调节机制，这对于维持细菌代谢很重要稳态。我们的发现提出了氧化还原介导的调节机制的假设，其中PGPA 通过控制谷胱甘肽介导的氧化还原平衡来调节细菌糖酵解的基于外部和内部的氧化还原平衡代谢信号。这种调节机制是新颖的，尚未在任何细胞类型中报道。进一步了解这种调节机制，我们将研究PGPA如何控制细菌葡萄糖摄取并调节使用生物化学，微生物学和代谢组方法的结合。了解PGPA如何调节细胞内氧化还原平衡，我们将检查谷胱甘肽生物合成和监视PGPA膜表面上的氧化还原变化，以证明PGPA如何使用综合性“ ying-- YANG”的机制可以达到代谢稳态和氧化还原平衡。我们还发现了氧化还原介导的 PGPA的调节是由PGPA二聚体内的二聚体二硫化物交联介导的。获得结构见解进入这种新型氧化还原调节的催化机制，我们将研究PGPA和副因素的催化活性 MG2+结合响应于使用生化测定的体外氧化还原变化的响应。我们还将研究这个分子使用FRET的机制来证明二聚体交联如何改变蛋白质对变构的构素更改活动位点构象以控制PGPA催化剂。由于没有结构 PGPA家族，我们将使用使用该态的两个不同的氧化还原（活跃/灭活）状态确定PGPA的结构 X射线晶体学和单粒子冷冻方法，以建立氧化还原的结构基础 PGPA的调节催化机制。这种机制在许多革兰氏阴性病原体中都是保守的。我们的研究将揭示一种了解微生物中代谢调节的重要机制。