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

• 批准号: 10652472
• 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
• 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/10652472
• 关键词: Active Sites; Anabolism; Antibiotic Resistance; 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; Cytoplasm; 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; Lipid Bilayers; Lipids; Measures; Mediating; Membrane; Metabolic; Metabolic Pathway; Metabolism; Methods; Microbiology; Molecular; Molecular Conformation; Monitor; Mutation; Oxidation-Reduction; 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; aminoacid biosynthesis; antimicrobial; bacterial metabolism; blood glucose regulation; cell growth; cell type; cofactor; crosslink; dimer; genetic approach; glucose uptake; inorganic phosphate; insight; metabolomics; microorganism; mutant; nanodisk; novel; novel therapeutic intervention; particle; pathogen; prevent; response; unnatural amino acids; vapor

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.

摘要 糖酵解是最重要的代谢途径之一，在真核生物和 原核生物。在这个过程中，葡萄糖被分解形成小的3-碳磷酸盐代谢物 细胞的生长和存活。在微生物中，适当地维持糖酵解对于发展 细菌感染与毒力和抗生素耐药性。在这个项目中，我们的目标是研究结构和 磷脂酰甘油磷酸酶PGPA在阐明糖酵解调节机制中的作用 细菌。PGPA是革兰氏阴性菌中普遍存在的一种完整的膜蛋白。我们发现 PGPA作为一种兼职酶起作用，即PGPA不仅参与磷脂的生物合成，而且还参与 作为一种重要的代谢调节剂，在E。 Coli.PGPA在大肠杆菌中的突变失活极大地促进了细菌的新陈代谢和生长。我们还有 发现了一种新的PGPA的氧化还原调节机制，它对维持细菌的代谢很重要 动态平衡。我们的发现提出了一种假设，即氧化还原介导的调节机制中，PGPA 通过控制谷胱甘肽介导的氧化还原平衡调节细菌糖酵解 代谢信号。这种调控机制是新颖的，目前还没有任何细胞类型的报道。为了进一步 了解了这种调节机制，我们将研究PGPA是如何控制细菌葡萄糖摄取和调节的 生物化学、微生物学和代谢组学相结合的糖酵解活性。 了解PGPA是如何调节细胞内氧化还原平衡的，我们将研究谷胱甘肽的生物合成和 监测PGPA膜表面氧化还原的变化，演示PGPA如何利用一体化的“鹰”- “阳”机制实现代谢动态平衡和氧化还原平衡。我们还发现了氧化还原介导的 PGPA的调节是通过PGPA二聚体中的二聚体二硫键交联来实现的。获得结构性洞察力 在这一新的氧化还原调节的催化机制中，我们将研究PGPA和辅因子的催化活性。 用生化分析方法研究镁离子结合对体外氧化还原反应的影响。我们也会研究这个分子 用FRET研究二聚体交联物改变蛋白质构象的机制 通过改变活性中心构象来控制PGPA的催化作用。中没有可用的结构。 作为PGPA家族的一员，我们将使用 X射线结晶学和单粒子低温电子显微镜方法，为氧化还原建立结构基础- 调节PGPA的催化机制。这一机制在许多革兰氏阴性杆菌中都是保守的。我们的 研究将揭示了解微生物代谢调节的重要机制。