MECHANISM OF KDO 8-P SYNTHASE

描述：当今医疗保健提供者面临的一个问题是 增加病原菌对经典抗菌药的耐药性。 克服这一问题的一个方法是设计新的和创新的 具有完全不同的行动模式的代理；因此，不 应出现与现有治疗药物的交叉耐药。多数 抗菌药物通过抑制生物合成中的关键酶起作用 微生物生存所必需的大分子分子。 这种方法的成功需要透彻地理解 在分子水平上的酶(S)。这项工作的目标是收集 3-脱氧-D-甘露八糖酶的作用机理 8-磷酸合成酶。在这些调查中收集到的信息 在未来选择性酶的设计和合成中应被证明是有用的 这种独特的酶的抑制剂，即新一代机械上的 多种革兰氏阴性抗生素。这个项目的具体目的是 3-脱氧-D-甘露八磺酸形成机理的建立 由阿拉伯糖5-磷酸(A5-P)和磷烯醇制得的8-磷酸(KDO8-P) KDO 8-P合成酶(EC 4.1.2.16)(KDO)催化的丙酮酸(PEP) 8-P S)，这种酶催化了 细菌脂多糖区A部分脂类的生物合成 革兰氏阴性细菌的细胞膜。定态和停流 将使用动力学来确定底物(S)结合的顺序和 产品发布，并提供对潜在产品的一些见解 核磁共振结构研究中间体(S)；多核核磁共振分析 KDO8-P合成酶与区域和立体特异性标记的相互作用 基材和产品的类似物，其中几种被设计成“可视化”潜力 瞬变中间体(S)和主动现场修饰使用 底物(S)/产物保护，结合定点突变 我们将利用这些研究进一步深入了解 各种酶对底物结合的功能及其作用机制 酶，特别是活性部位的半胱氨酸。X射线结晶学 研究将提供有关星体的三维结构的信息 固态的酶。实验策略旨在 允许探索几种机械的可能性。

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.