Salicylate production in siderophore biosynthesis

铁载体生物合成中水杨酸的产生

• 批准号: 8091456
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8091456
• 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）：铁载体是由细菌产生的低分子量铁螯合剂，用于从环境中清除铁，并且经常需要细菌毒力。在限制铁的环境中，如人类宿主，如果铁载体的生产被破坏，细菌就不能获得生长和生存所需的铁。众所周知，铜绿假单胞菌具有抗生素耐药性，能够在免疫功能低下的患者和囊性纤维化患者的肺部定植。本实验室研究的长期目标是提供铁载体生物合成途径中酶的详细机制和结构理解，这些途径是药物发现的目标。为此，我们建议研究产生水杨酸的非核糖体肽合成酶辅助酶，水杨酸是细菌P. aeruginosa中铁载体pyochelin的前体。所研究的两种酶，即异丙酸酯合成酶（PchA）和异丙酸酯裂解酶（PchB），是在理解催化反应途径方面取得根本性进展的理想模型系统。我们将使用多方面的方法，包括酶动力学分析，结构生物学和计算酶学，并通过高通量筛选确定酶的抑制剂。我们的第一个具体目标将是确定活性底物构象和静电过渡态稳定在由具有外源性氯酸突变酶活性的异丙酸-丙酮酸裂解酶进行的周环反应机制中的相对催化作用。在我们的第二个目标中，我们将定义MST蛋白家族成员Mg+2依赖性异choris酸合成酶的结构-功能关系。最后，我们的第三个目标是确定水杨酸生产的小分子抑制剂，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（包括接受化疗的癌症患者、艾滋病患者、免疫缺陷患者、囊性纤维化患者、烧伤患者和包括婴儿在内的其他危险个体）感染的常见原因。这项工作的目的是提供对铜绿假单胞菌中促进毒力的两种酶的基本了解，以便利用这些信息来产生新的抗菌药物。