ENZYMOLOGY OF ALGINATE BIOSYNTHESIS

描述：（申请人摘要中的逐字记录）：拟定研究 该计划是一个详细的调查到酶的海藻酸盐 在病原性细菌铜绿假单胞菌中的生物合成。铜绿假单胞菌 感染是常见的，并且对人类的健康造成重大危害。 铜绿假单胞菌在肺组织定植引起的并发症是 囊性纤维化患者发病率和死亡率的主要原因。 海藻酸盐是由甘露糖醛酸和古洛糖醛酸组成的线型多糖 残基，并且由细菌分泌以形成胞外囊， 这有助于它们有效地定植在肺组织中， 抗生素治疗和逃避宿主的免疫系统反应。一个潜在 对抗铜绿假单胞菌感染的策略是开发抑制 藻酸盐的生物合成，从而使细菌对 常规抗生素。文献中的报告表明，这种策略 具有优点，但迄今为止，没有有效的特异性抑制剂或 铜绿假单胞菌藻酸盐生物合成酶的灭活剂。 为了合理探讨海藻酸盐生物合成的抑制作用， 更深入地了解的功能特性和催化机制， 所述途径的组成酶是必需的。研究计划 在提案中描述的重点是C5甘露糖醛酸差向异构酶和酶 其催化藻酸盐生物合成途径的前四步。 GDP-甘露糖脱氢酶催化藻酸盐中的关键步骤 生物合成，一种有趣的四电子氧化机制， 受到特别严格的审查。使用瞬态的详细动力学研究 动力学方法和动力学同位素效应测量将在 以确定反应的能量学;潜在的抑制剂和 灭活剂是根据酶的 化学机制将被描述。磷酸甘露变位酶的结构， 催化第二步反应的物质将由X射线测定 结晶学; GDP-甘露糖脱氢酶也已结晶， 将继续确定其结构。

DESCRIPTION: (Verbatim from the Applicant's Abstract): The proposed research program is a detailed investigation into the enzymology of alginate biosynthesis in the pathogenic bacterium Pseudomonas aeruginosa. P. aeruginosa infections are common and present significant health hazards to humans. Complications arising from colonization of lung tissues by P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis patients. Alginate is a linear polysaccharide composed of mannuronate and guluronate residues, and is secreted by the bacteria to form an extracellular capsule, which contributes to their ability to effectively colonize lung tissue, resist antibiotic therapies and evade the host's immune system response. A potential strategy to combat P. aeruginosa infections is to develop agents which inhibit alginate biosynthesis and thereby render the bacteria susceptible to conventional antibiotics. Reports in the literature suggest that this strategy has merit, but to date, there are no effective specific inhibitors or inactivators of P. aeruginosa alginate biosynthetic enzymes. In order to approach the inhibition of alginate biosynthesis in a rational way, a deeper understanding of the functional properties and catalytic mechanisms of the constituent enzymes of the pathway is required. The research program described in the proposal focuses on C5 mannuronan epimerase and the enzymes which catalyze the first four steps of the alginate biosynthetic pathway. GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis, a mechanistically interesting four-electron oxidation, and will receive particularly close scrutiny. Detailed kinetic studies using transient kinetic approaches and kinetic isotope effect measurements will be performed in order to determine the energetics of the reactions; potential inhibitors and inactivators which have been designed based on hypotheses about the enzyme's chemical mechanisms will be characterized. The structure of phosphomannomutase, which catalyzes the second step in the pathway, will be determined by X-ray crystallography; GDP-mannose dehydrogenase has also been crystallized, and the determination of its structure will be pursued.