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：
10280369
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/10280369
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.

抽象的糖酵解是真核生物和生物中最重要的代谢途径之一。在原核生物中，葡萄糖被分解形成小分子三碳磷酸盐代谢物。在微生物中，适当维持糖酵解对于细胞的生长和存活非常重要。在这个项目中，我们的目标是研究细菌感染和毒力以及抗生素耐药性。磷脂酰甘油磷酸酶 PgpA 的功能阐明糖酵解的新调节机制 PgpA 是一种普遍存在于革兰氏阴性细菌中的膜蛋白。 PgpA 作为兼职酶发挥作用；即 PgpA 不仅参与磷脂生物合成，还参与磷脂生物合成。通过水解大肠杆菌中关键的 3-碳磷酸糖酵解代谢物，充当重要的代谢调节剂。大肠杆菌中 PgpA 的突变失活也极大地促进了细菌的代谢和生长。确定了 PgpA 的一种新的氧化还原调节机制，这对于维持细菌代谢很重要我们的研究结果提出了 PgpA 介导的氧化还原调节机制的假设。通过控制基于外部和内部的谷胱甘肽介导的氧化还原平衡来调节细菌糖酵解这种调节机制是新颖的，尚未在任何细胞类型中得到进一步报道。了解了这种调节机制，我们将研究PgpA如何控制细菌葡萄糖摄取并调节结合生物化学、微生物学和代谢组学方法来检测糖酵解活性。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测 PgpA 膜表面的氧化还原变化，以演示 PgpA 如何使用综合“Ying- Yang”机制同时实现代谢稳态和氧化还原平衡我们还发现了氧化还原介导的机制。 PgpA 的调节是由 PgpA 二聚体内的二聚体二硫键交联介导的。为了研究这种新颖的氧化还原调节催化机制，我们将研究 PgpA 和辅因子的催化活性使用生化测定来响应体外氧化还原变化的 Mg2+ 结合我们还将研究这种分子测定。使用 FRET 的机制来演示二聚体交联如何将蛋白质构象改变为变构由于没有结构可用，因此改变活性位点构象以控制 PgpA 催化。 PgpA 家族，我们将使用以下方法确定两种不同氧化还原（活性/失活）状态下的 PgpA 结构： X 射线晶体学和单粒子冷冻电镜方法为氧化还原-建立结构基础 PgpA 的调节催化机制在许多革兰氏阴性病原体中是保守的。研究将揭示理解微生物代谢调节的重要机制。