ENZYMOLOGY OF ALGINATE BIOSYNTHESIS

描述：(逐字摘自申请人的摘要)：拟议的研究 计划是对海藻酸酶学的详细调查 铜绿假单胞菌的生物合成。铜绿假单胞菌 感染是常见的，对人类的健康构成重大威胁。 铜绿假单胞菌在肺组织定植引起的并发症有 囊性纤维化患者的发病率和死亡率的主要原因。 海藻酸盐是一种由甘露酸和古鲁酸组成的线性多糖。 残留物，由细菌分泌形成胞外包膜， 这有助于它们有效地在肺组织上定植，抵抗 抗生素疗法和逃避宿主的免疫系统反应。一种潜力 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，这一策略 有优点，但到目前为止，还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。 为了合理地探讨抑制藻酸盐生物合成的方法， 对其功能性质和催化机理有更深入的了解 该途径的组成酶是必需的。研究计划 提案中描述的重点是C5甘露聚糖表异构酶和酶 它们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承诺步骤 生物合成，一种机械上有趣的四电子氧化，并将 受到特别严密的审查。使用瞬变法进行详细的动力学研究 动力学方法和动力学同位素效应测量将在#年进行。 以确定反应的能量学；潜在的抑制剂和 根据酶的假说设计的灭活剂 将对化学机制进行表征。磷酸甘露糖变位酶的结构， 途径中的第二步是什么催化的，将通过X射线来确定 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.