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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：
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
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/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

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.

摘要