Salicylate production in siderophore biosynthesis

铁载体生物合成中水杨酸的产生

• 批准号: 8288743
• 负责人: AUDREY L LAMB
• 金额: $ 28.81万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288743
• 关键词: AIDS chemotherapy; Acids; Active Sites; Adopted; Affinity; Anabolism; Antibiotic Resistance; Bacteria; Binding; Biological Models; Burn injury; Cancer Patient; Catalysis; Chelating Agents; Chorismate Mutase; Crystallography; DNA Sequence Rearrangement; Dependence; Drug Design; Electrostatics; Environment; Enzymatic Biochemistry; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Family; Free Energy; Goals; Growth; Health; Human; Immune; Individual; Infant; Infection; Iron; Isotopes; Kinetics; Laboratories; Lead; Link; Lung; Lyase; Molecular; Molecular Conformation; Molecular Weight; Motion; Mutation; Pathogenesis; Pathway interactions; Patients; Production; Property; Protein Family; Pseudomonas aeruginosa; Publishing; Pyruvate; Quantum Mechanics; Reaction; Relative (related person); Research; Risk; Role; Siderophores; Solvents; Structure; Structure-Activity Relationship; Testing; Thermodynamics; Vertebral column; Virulence; Work; antimicrobial drug; base; cystic fibrosis patients; drug discovery; enzyme mechanism; high throughput screening; inhibitor/antagonist; innovation; insight; member; molecular dynamics; molecular mechanics; pathogen; peptide synthase; pyochelin; salicylate; small molecule; structural biology

DESCRIPTION (provided by applicant): Siderophores are low molecular weight iron chelators produced by bacteria to scavenge iron from the environment, and are frequently required for bacterial virulence. In iron limiting environments such as the human host, if the production of the siderophore is disrupted, the bacteria cannot acquire the iron required for growth and survival. Pseudomonas aeruginosa are notoriously antibiotic-resistant and are able to colonize immuno-compromised patients and the lungs of patients with cystic fibrosis. The long-term goal of the research in this laboratory is to provide a detailed mechanistic and structural understanding of enzymes in the siderophore biosynthetic pathways that are targets for drug discovery. Toward that end, we propose to investigate the nonribosomal peptide synthetase accessory enzymes that produce salicylate, a precursor incorporated into the siderophore pyochelin from the bacteria P. aeruginosa. The two enzymes to be studied, the isochorismate synthase (PchA) and isochorismate-pyruvate lyase (PchB), are ideal model systems for making fundamental advances in understanding the reaction pathways of catalysis. We will use a multifaceted approach that includes enzyme kinetic analysis, structural biology and computational enzymology, and identify inhibitors of the enzymes through high-throughput screening. Our first specific aim will be to determine the relative catalytic contributions of a reactive substrate conformation and electrostatic transition state stabilization in the pericyclic reaction mechanisms performed by the isochorismate- pyruvate lyase that has adventitious chorismate mutase activity. We will define the structure- function relationships of the Mg+2 dependent isochorismate synthase, a member of the MST family of proteins in our second aim. Finally, our third aim is to identify small molecule inhibitors of salicylate production, and determine their mode of action. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a dangerous opportunistic pathogen that is a common cause of infections in susceptible hosts, including cancer patients undergoing chemotherapy, AIDS patients, those with immune deficiencies, cystic fibrosis patients, burn patients and other at risk individuals including infants. The goal of this work is to provide a fundamental understanding of two enzymes that promote virulence in P. aeruginosa so that this information can be exploited to generate new antimicrobial drugs.

描述（由申请人提供）：铁载体是细菌生产的低分子量铁螯合剂，以从环境中清除铁，并且经常需要细菌毒力。在铁限制环境（例如人类宿主）中，如果辅助载体的产生被破坏，细菌将无法获得生长和生存所需的铁。铜绿假单胞菌是众所周知的抗生素耐药性，能够在免疫受损的患者和囊性纤维化患者的肺部定植。该实验室研究的长期目标是在铁载体生物合成途径中提供详细的机械和结构理解，这是用于药物发现的目标。为此，我们建议研究产生水杨酸盐的非核糖体肽合成酶辅助酶，这是一种从铜绿假单胞菌中掺入侧酚毕胆素中的前体。要研究的两种酶，同义合成酶（PCHA）和等化型 - 丙酮酸裂解酶（PCHB）是理想的模型系统，用于在理解催化反应途径方面取得了基本进步。我们将使用包括酶动力学分析，结构生物学和计算酶学的多方面方法，并通过高通量筛选鉴定酶的抑制剂。我们的第一个具体目的是确定反应性底物构象和静电过渡状态稳定在具有不定层合基酸突变酶活性的同型 - 丙酮酸裂解酸酯裂解液中的生周期反应机制中的相对催化作用。我们将定义Mg+2依赖性等齿合酶的结构 - 功能关系，这是我们第二个目标中MST蛋白质家族的成员。最后，我们的第三个目标是鉴定出水杨酸酯产生的小分子抑制剂，并确定其作用方式。公共卫生相关性：铜绿假单胞菌是一种危险的机会性病原体，是易感宿主感染的常见原因，包括接受化学疗法的癌症患者，艾滋病患者，免疫缺陷患者，囊性纤维化患者，烧伤患者，烧伤患者，包括婴儿在内的其他患者。这项工作的目的是提供对促进铜绿假单胞菌毒力的两种酶的基本理解，以便可以利用此信息来生成新的抗菌药物。