Mechanism of A5P Isomerase

A5P异构酶的机制

• 批准号: 7559583
• 负责人: Ronald Wesley Woodard
• 金额: $ 36.2万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7559583
• 关键词: 5' -AMP-activated protein kinase; Acids; Active Sites; Address; Affect; Amino Acids; Anabolism; Antibiotics; Arabinose-5-phosphate isomerase; Aspartate; Binding; Biochemical; Biological Assay; Blast Cell; C-terminal; Catalysis; Cell physiology; Cells; Characteristics; Chimera organism; Chimeric Proteins; Chloride Channels; Complement component C1s; Cystathionine; Cystathionine beta-Synthase; Cytidine Monophosphate; D-arabitol; D-arabitol 5-phosphate; Data; Discrimination; Endotoxins; Enzymes; Equilibrium; Escherichia; Escherichia coli; Exhibits; Family; Figs - dietary; Gene Proteins; Generations; Genes; Genomics; Glucose; Glucose-6-Phosphate; Glutamates; Glycerophospholipids; Goals; Gram-Negative Bacteria; Grant; Hydrogen; Hydroxy Acids; In Vitro; Individual; Inosine Monophosphate; Ion Transport; Isomerase; Isomerase Gene; K antigen; Ketoses; Ketosis; Kinetics; Knock-out; Label; Laboratory Study; Length; Libraries; Lipopolysaccharides; Mannose; Mediating; Membrane; Metabolism; Methodology; Modification; Monosaccharides; Open Reading Frames; Operon; Organism; Oxidoreductase; Oxygen; Pathway interactions; Pentoses; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferase Gene; Phosphotransferases; Physiological; Polysaccharides; Post-Translational Regulation; Principal Investigator; Production; Protein Family; Proteins; Protons; Reagent; Regulation; Regulon; Research Personnel; Resistance; Ribose-5-phosphate isomerase; Role; Rosa; Screening procedure; Site-Directed Mutagenesis; Solvents; Sorbitol; Source; Substrate Specificity; System; Techniques; Testing; Trioses; Validation; Virulence; X ray diffraction analysis; X-Ray Diffraction; analog; anomer; arabinose 5-phosphate; asparaginase; base; capsule; dehydrogenation; deprotonation; design; expression vector; genetic regulatory protein; high throughput screening; inhibitor/antagonist; inorganic phosphate; insight; macromolecule; maltose dehydrogenase; mannose 6 phosphate; member; microorganism; monomer; novel; numb protein; preference; programs; proton-translocating pyrophosphatase; protonation; ribose-5-phosphate; ribulose 5-phosphate; small molecule; stem; stereochemistry; sugar; sugar nucleotide; thermophilic organism; three dimensional structure; voltage; web site

This grant proposes to collect mechanistic information on and understand the regulation of D-arabinose 5- phosphate (ASP) isomerase (API), the enzyme responsible for the synthesis of ASP from D-ribulose 5- phosphate (Ru5P), in Gram-(-) microorganisms. This information should provide validation that API, a key enzyme in lipopolysaccharide (LPS-aka endotoxin) biosynthesis, is a target for screening efforts by investigators in the field to identify selective inhibitors of API - namely a new generation ofmechanistically diverse antibiotics for which no resistance is known. The goals of this project are to establish 1. the intracellular function of the ASP isomerases expressed by the genes yrbH, kpsF, c3406 and gutQ as well as to understand the regulatory role of the cystathionine beta -synthase domain (CBS) common to three of the gene products; 2. the mechanism for the formation of ASP from RuSP; and 3. the substrate specificity of the APIs and the role of active site amino acids. The specific aims focus on diverse techniques to detect the presence of an intermediate enediol and to determine the stereochemistry of the inter-conversion of the substrate and product. Genomic knockouts will be constructed to understand the cellular function of each API as well as the potential lethality of a genomic knockout of API (target validation). Truncated derivatives of API lacking the CBS domain will be utilized to ascertain CBS domain function and a library of sugar nucleotides will be screened to find potential regulators of the CBS domain. Site-directed mutagenesis studies, based on crystallographic data and active site modification using mechanism-based irreversible inhibitors, will be exploited to gain insight into the contribution of enzyme functionalities to substrate binding, monomer interface interactions, and to the mechanism of API. The small molecules to be synthesized in this grant will serve as mechanistic probes. The ultimate goal of these studies is to better understand the role of LPS, a critical macromolecule essential to both the survival and virulence of Gram-(-) microorganisms

这项拨款旨在收集有关d -阿拉伯糖5-调控的机制信息和理解