MECHANISM OF KDO 8-P SYNTHASE

KDO 8-P 合成酶的机制

• 批准号: 2734772
• 负责人: Ronald Wesley Woodard
• 金额: $ 18.86万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734772
• 关键词: X ray crystallography; active sites; aldehyde lyase; arabinose; bacterial polysaccharides; chemical kinetics; enzyme mechanism; enzyme structure; enzyme substrate complex; isozymes; microorganism metabolism; nuclear magnetic resonance spectroscopy; phosphoenolpyruvate; site directed mutagenesis

DESCRIPTION: A problem confronting present day health care providers is an increasing resistance in pathogenic bacteria to the classic antibacterials. An approach to overcoming this problem is to design new and innovative agents with a totally different mode of action; therefore, no cross-resistance with present therapeuticals should occur. Most antimicrobial drugs act by inhibiting key enzymes in the biosynthesis of macromolecular molecules necessary for viability of the microorganism. Success in this type of approach necessitates a thorough understanding of the enzyme(s) at the molecular level. The goal of this work is to collect mechanistic information about the enzyme 3-deoxy-D-manno-octulosonic 8-phosphate synthase. The information collected in these investigations should prove useful in the future design and synthesis of selective enzyme inhibitors of this unique enzyme, namely a new generation of mechanistically diverse gram negative antibiotics. The specific aim of this project is to establish the mechanism for the formation of 3-deoxy-D-manno-octulosonic 8-phosphate (KDO 8-P) from arabinose 5-phosphate (A 5-P) and phosphoenol pyruvate (PEP) catalyzed by the enzyme KDO 8-P synthase (EC 4.1.2.16) (KDO 8-P S), the enzyme which catalyzes one of the first committed steps in the biosynthesis of the lipid A portion of the lipopolysaccharide region of the cell envelope of gram-negative bacteria. Steady-state and stopped-flow kinetics will be used to determine the order of substrate(s) binding and product release as well as provide some insight into the potential intermediate(s) for NMR structural studies; multinuclear NMR analysis of the interaction of KDO 8-P synthase with regio- and stereo-specific labeled substrate and product analogs, several designed to "visualize" the potential transient intermediate(s), and active site modification employing substrate(s)/product protection, coupled with site-directed mutagenesis studies will be exploited to gain further insight into the contribution of various enzyme functionalities to substrate binding and the mechanism of the enzyme, in particular active site cysteines. An X-ray crystallographic study will provide information concerning the three-dimensional structure of the enzyme in the solid state. The experimental strategies are designed to allow probing of several mechanistic possibilities.

描述：当今卫生保健提供者面临的一个问题是， 增加病原菌对经典抗菌药物的耐药性。 解决这个问题的一个方法是设计新的和创新的 具有完全不同作用模式的药剂;因此，没有 与现有治疗剂的交叉耐药性将会发生。 最 抗微生物药物通过抑制生物合成中的关键酶起作用， 微生物存活所必需的大分子。 这种方法的成功需要彻底了解 分子水平上的酶。 这项工作的目标是收集 有关3-脱氧-D-甘露糖-八酮糖酶的机械信息 8-磷酸合酶 在这些调查中收集的信息 在未来的选择性酶的设计和合成中应该是有用的 这种独特酶的抑制剂，即新一代的机械抑制剂 多种革兰氏阴性抗生素 该项目的具体目标是 建立了3-脱氧-D-甘露-辛酮糖酸的形成机制 8-从阿拉伯糖5-磷酸（A5-P）和磷酸烯醇得到的磷酸（KDO 8-P） 由酶KDO 8-P合酶（EC 4.1.2.16）（KDO）催化的丙酮酸（PEP 8-PS），该酶催化的第一个承诺的步骤之一， 在一些实施方案中，所述方法包括使脂多糖区域的脂质A部分生物合成， 革兰氏阴性菌的细胞被膜。 稳态和停流 动力学将用于确定底物结合的顺序， 产品发布以及提供一些洞察的潜力， NMR结构研究的中间体; KDO 8-P合酶与区域和立体特异性标记相互作用 底物和产物类似物，一些设计用于“可视化”潜在的 瞬时中间体和活性位点修饰， 底物/产物保护，结合定点诱变 研究将被利用，以进一步了解的贡献， 各种酶的功能，底物结合和机制， 酶，特别是活性位点半胱氨酸。 X射线晶体学 研究将提供有关三维结构的信息， 固态的酶。 实验策略旨在 允许探测几种机械可能性。