Salicylate production in siderophore biosynthesis

说明(申请人提供)：铁载体是由细菌产生的低分子铁络合剂，用于从环境中清除铁，通常是细菌毒力所必需的。在人类宿主等铁限制环境中，如果铁载体的生产受到干扰，细菌就无法获得生长和生存所需的铁。众所周知，铜绿假单胞菌对抗生素具有抗药性，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。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的酶的详细机制和结构的了解，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盐的非核糖体多肽合成酶辅助酶，水杨酸盐是一种从铜绿假单胞菌的铁载体蛋白球蛋白中并入的前体。将被研究的两种酶，即异分枝酸合成酶(PchA)和异分枝酸-丙酮酸裂解酶(PchB)，是在理解催化反应途径方面取得基础进展的理想模型系统。我们将使用包括酶动力学分析、结构生物学和计算酶学在内的多方面方法，通过高通量筛选来识别酶的抑制剂。我们的第一个特定目标将是确定具有不定分支酸变位酶活性的异分支酸-丙酮酸裂解酶在周环反应机制中反应底物构象和静电过渡态稳定的相对催化作用。在我们的第二个目标中，我们将定义MST蛋白家族中的一个成员，依赖于镁的异分支酸合成酶的结构与功能的关系。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，并确定它们的作用模式。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、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高危个人。这项工作的目标是提供对两种促进铜绿假单胞菌毒力的酶的基本了解，以便利用这些信息来生产新的抗微生物药物。

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.