BIOSYNTHESIS OF BACTERIAL ALGINATE

细菌藻酸盐的生物合成

• 批准号: 2185104
• 负责人: JOHN M BEALE
• 金额: $ 11.07万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185104
• 关键词: Azotobacter vinelandii; Pseudomonas aeruginosa; active sites; alginates; carbohydrate biosynthesis; chemical structure function; enzyme mechanism; enzyme substrate complex; isomerase; metal complex; nuclear magnetic resonance spectroscopy; polymerization; stable isotope

The long term goal of this research project is to thoroughly define the biosynthetic pathway of alginate biosynthesis, in terms of its individual steps and enzymology. These studies will lead, eventually to inhibitors for enzymes of the pathway that may be useful as drugs in the treatment of mucoid Pseudomonas infections in cystic fibrosis patients. During the period of the proposed work, we will focus our efforts on several key portions of the pathway: mannuronan C5-epimerase, GDP-manuronate polymerase, the alginate acetylation steps, and the early metabolite fluxes into the alginate pathway. These studies will serve as a foundation for future design of pathway-specific inhibitors and for sequencing and structure determination of the enzymes. Studies will in all cases by comparative between Pseudomonas aeruginosa and the prototype alginate producer, Azotobacter vinelandii. Our specific aims, listed in order of priority, during this funding period will be: (1) Isolation, purification, and characterization of mannuronan C5- epimerase from Azotobacter vinelandii and Pseudomonas aeruginosa (cystic fibrosis isolates), and conducting initial studies of its active site. (2) Studies of internal isotope return in the epimerase to determine whether a one- or two-base catalytic mechanism is operating. (3) Further development of our model for polymer-level control of epimerase action pattern, including binding studies in the presence of various metal ions. (4) NMR/molecular modeling studies of metal ion complexes with alginate components to elucidate conformational dictates of the epimerase. (5) Elucidate the functions of the O2 and O3 acetyl substituents in bacterial alginate. (6) Elucidate the location and polymerization mechanism of GDP-mannuronate polymerase, and identify the initiation carrier molecule. (7) Survey, through stable isotope/NMR methods, primary metabolic fluxes feeding into the alginate pathway.

本研究项目的长期目标是彻底定义 藻酸盐生物合成的生物合成途径，就其个体而言， 步骤和酶学。 这些研究最终将导致抑制剂 对于可用作治疗以下疾病的药物的途径的酶， 囊性纤维化患者的粘液假单胞菌感染。 期间 在建议的工作期间，我们将集中精力在几个关键方面 途径的部分：甘露糖醛酸C5-差向异构酶，GDP-甘露糖醛酸 聚合酶、藻酸盐乙酰化步骤和早期代谢物通量 进入藻酸盐通道。 这些研究将作为基础， 途径特异性抑制剂的未来设计和测序， 酶的结构测定。 研究将在所有情况下， 铜绿假单胞菌与原型藻酸盐的比较 生产者棕色固氮菌（Azotobacter vinelandii）。 我们在本资助期内的具体目标，按优先次序列出 将为： (1)甘露糖醛酸C5- 来自棕色固氮菌和铜绿假单胞菌（囊性）的差向异构酶 纤维化分离株），并对其活性位点进行初步研究。 (2)差向异构酶中的内部同位素返回研究 无论是单基催化机制还是双基催化机制在运行。 (3)差向异构酶聚合物水平控制模型的进一步发展 作用模式，包括在各种金属存在下的结合研究 离子。 (4)海藻酸金属离子配合物的NMR/分子模拟研究 组分以阐明差向异构酶的构象指示。 (5)阐明O2和O3乙酰基取代基在 细菌藻酸盐 (6)阐明了GDP-甘露糖醛酸的位置和聚合机理 聚合酶，并鉴定起始载体分子。 (7)通过稳定同位素/核磁共振方法调查初级代谢通量 进入藻酸盐通道。