Mechanism of A5P Isomerase

A5P异构酶的机制

• 批准号: 7169212
• 负责人: Ronald Wesley Woodard
• 金额: $ 36.9万
• 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/7169212
• 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; Numbers; 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

DESCRIPTION (provided by applicant): 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 of mechanistically 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

描述（由申请人提供）：本授权计划收集有关D-阿拉伯糖5-磷酸（ASP）异构酶（API）的机制信息并了解其调节，该酶负责在革兰氏（-）微生物中从D-核酮糖5-磷酸（Ru 5 P）合成ASP。该信息应提供以下验证：API（脂多糖（LPS-又名内毒素）生物合成中的关键酶）是本领域研究人员筛选工作的目标，以鉴定API的选择性抑制剂-即新一代机制多样的抗生素，已知无耐药性。该项目的目标是建立1。由基因yrbH、kpsF、c3406和gutQ表达的ASP异构酶的细胞内功能，以及理解三种基因产物共有的胱硫醚β-合酶结构域（CBS）的调节作用; 2. RuSP生成ASP的机理; 3. API的底物特异性和活性位点氨基酸的作用。具体目标集中在不同的技术，以检测中间体的存在，并确定底物和产物的相互转化的立体化学。将构建基因组敲除，以了解每种API的细胞功能以及API基因组敲除的潜在致死性（靶标验证）。将利用缺少CBS结构域的API的截短衍生物来确定CBS结构域功能，并筛选糖核苷酸文库以发现CBS结构域的潜在调节剂。基于晶体学数据和使用基于机制的不可逆抑制剂的活性位点修饰的定点诱变研究，将被用来深入了解酶功能对底物结合、单体界面相互作用和API机制的贡献。在这项资助下合成的小分子将作为机械探针。这些研究的最终目的是更好地了解LPS的作用，LPS是革兰氏（-）微生物生存和毒力所必需的关键大分子