ENZYMOLOGY OF ALGINATE BIOSYNTHESIS

海藻酸盐生物合成的酶学

• 批准号: 6127412
• 负责人: Peter A Tipton
• 金额: $ 21万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6127412
• 关键词: Pseudomonas aeruginosa; X ray crystallography; alcohol oxidoreductases; alginates; bioenergetics; carbohydrate biosynthesis; drug resistance; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; phosphomutase

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.

描述：（逐字摘自申请人摘要）：拟议的研究 计划是对海藻酸盐酶学的详细研究 致病菌铜绿假单胞菌中的生物合成。铜绿假单胞菌 感染很常见，对人类健康构成重大危害。 铜绿假单胞菌定植肺组织引起的并发症是 囊性纤维化患者发病和死亡的主要原因。 海藻酸盐是由甘露糖醛酸和古洛糖醛酸组成的线性多糖 残留物，并由细菌分泌形成细胞外囊， 这有助于它们有效定植肺组织、抵抗 抗生素疗法并逃避宿主的免疫系统反应。有潜力 对抗铜绿假单胞菌感染的策略是开发抑制铜绿假单胞菌感染的药物 藻酸盐生物合成，从而使细菌易受 常规抗生素。文献报道表明该策略 有其优点，但迄今为止，尚无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。 为了以合理的方式解决海藻酸盐生物合成的抑制问题， 更深入地了解其功能特性和催化机制 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 甘露糖醛差向异构酶和酶 它催化藻酸盐生物合成途径的前四个步骤。 GDP-甘露糖脱氢酶催化海藻酸盐中的关键步骤 生物合成，一种机械上有趣的四电子氧化，并且将 受到特别严格的审查。使用瞬态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 以确定反应的能量；潜在的抑制剂和 灭活剂是根据酶的假设而设计的 化学机制将得到表征。磷酸甘露糖变位酶的结构， 催化途径的第二步，将通过 X 射线测定 晶体学； GDP-甘露糖脱氢酶也已结晶， 将继续确定其结构。