Mechanism of A5P Isomerase

A5P异构酶的机制

• 批准号: 7749996
• 负责人: Ronald Wesley Woodard
• 金额: $ 35.84万
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749996
• 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

这项资助旨在收集有关D-阿拉伯糖5-磷酸化的机制信息，并了解其调控机制。 磷酸（ASP）异构酶（API），负责从D-核酮糖5- 磷酸盐（Ru 5 P），在革兰氏（-）微生物中。此信息应提供API（密钥） 脂多糖（LPS-aka内毒素）生物合成中的酶，是通过 本领域的研究人员鉴定了API的选择性抑制剂-即新一代的机械抑制剂 多种抗生素，已知没有耐药性。该项目的目标是建立1。的 由基因yrbH、kpsF、c3406和gutQ表达的ASP异构酶的细胞内功能，以及 为了了解胱硫醚β-合酶结构域（CBS）的调节作用， 基因产物; 2. RuSP生成ASP的机理; 3.的底物专一性 活性位点氨基酸的作用。具体目标集中在不同的技术来检测 存在的中间engly和确定立体化学的相互转化， 基质和产品。将构建基因组敲除以了解每个基因的细胞功能。 API以及API基因敲除的潜在致死性（靶标验证）。截断导数 将利用缺乏CBS结构域的API的100%来确定CBS结构域功能和糖库 将筛选核苷酸以发现CBS结构域的潜在调节剂。定点诱变 研究，基于晶体学数据和活性位点修饰，使用基于机制的不可逆 抑制剂，将被利用来深入了解酶功能对底物结合的贡献， 单体界面相互作用，以及API的作用机理。在这个过程中要合成的小分子 格兰特将作为机械探针。这些研究的最终目标是更好地了解 LPS是革兰氏（-）微生物生存和毒力的关键大分子

项目成果

A novel glucose 6-phosphate isomerase from Listeria monocytogenes.

一种来自单核细胞增生李斯特菌的新型葡萄糖 6-磷酸异构酶。

• DOI: 10.1007/s10930-014-9577-7
• 发表时间: 2014
• 期刊: The protein journal
• 作者: Cech,DavidL;Wang,Pan-Fen;Holt,MelissaC;Assimon,VictoriaA;Schaub,JeffreyM;Holler,TodP;Woodard,RonaldW
• 通讯作者: Woodard,RonaldW

Enediol mimics as inhibitors of the D-arabinose 5-phosphate isomerase (KdsD) from Francisella tularensis.

Enediol 模拟土拉弗朗西斯菌 D-阿拉伯糖 5-磷酸异构酶 (KdsD) 的抑制剂。

• DOI: 10.1016/j.bmcl.2010.12.066
• 发表时间: 2011
• 期刊: Bioorganic & medicinal chemistry letters
• 影响因子: 2.7
• 作者: Yep,Alejandra;Sorenson,RoderickJ;Wilson,MichaelR;Showalter,HDHollis;Larsen,ScottD;Keller,PaulR;Woodard,RonaldW
• 通讯作者: Woodard,RonaldW

Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.

对脆弱拟杆菌的阿拉伯糖 5-磷酸异构酶的分析提供了对单域阿拉伯糖磷酸异构酶调节的深入了解。

• DOI: 10.1128/jb.01735-14
• 发表时间: 2014
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Cech,David;Wang,PanFen;Holler,TodP;Woodard,RonaldW
• 通讯作者: Woodard,RonaldW

A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073.

在基因组岛中发现的一种独特的阿拉伯糖 5-磷酸异构酶，与大肠杆菌 CFT073 的尿路致病性相关。

• DOI: 10.1128/jb.00033-11
• 发表时间: 2011
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Mosberg,JoshuaA;Yep,Alejandra;Meredith,TimothyC;Smith,Sara;Wang,Pan-Fen;Holler,TodP;Mobley,HarryLT;Woodard,RonaldW
• 通讯作者: Woodard,RonaldW