Salicylate production in siderophore biosynthesis

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，用于从环境中吸收铁，并且通常是细菌毒力所需的。在铁限制的环境中，如人类宿主，如果铁载体的产生被破坏，细菌就不能获得生长和生存所需的铁。铜绿假单胞菌是出了名的耐药性，并且能够定殖免疫受损的患者和患有囊性纤维化的患者的肺。该实验室研究的长期目标是提供对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，水杨酸是从细菌铜绿假单胞菌中掺入铁载体绿脓菌螯铁蛋白的前体。待研究的两种酶，异分支酸合酶（PchA）和异分支酸丙酮酸裂解酶（PchB），是理解催化反应途径的根本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的反应底物构象和静电过渡态稳定的周环反应机制进行的异分支酸-丙酮酸裂解酶，具有外源分支酸裂解活性的相对催化贡献。在我们的第二个目标中，我们将定义Mg+2依赖性异分支酸合酶的结构-功能关系，该合酶是MST蛋白家族的成员。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，是易感宿主感染的常见原因，包括接受化疗的癌症患者、AIDS患者、免疫缺陷患者、囊性纤维化患者、烧伤患者和其他高危个体，包括婴儿。这项工作的目标是提供一个基本的了解两种酶，促进铜绿假单胞菌的毒力，使这些信息可以被利用来产生新的抗菌药物。

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.