Mechanism of KDO 8-P and DAH 7-P Synthase

描述：（由申请人提供）卫生保健提供者每天都受到挑战 病原菌对抗菌药物的耐药性增加， 阿森纳官方要克服这一问题，就必须设计新的和创新的 具有完全不同作用模式的抗生素， 目前的代理商应该会出现。大多数抗微生物药物通过抑制 生物合成大分子所必需的关键酶， 微生物的生存能力。这种方法的成功需要 在分子水平上彻底了解酶。的目标 这项工作是收集酶的机理信息， 3-脱氧-D-甘露-辛酮糖酸8-磷酸和3-脱氧-D-阿拉伯-庚酮糖酸 7-磷酸合酶这些信息将被证明是有用的设计 这些独特酶的选择性抑制剂，即新一代的 不同的抗生素。该项目的目标是建立 1. 3-脱氧-D-甘露-辛酮糖-8-磷酸的形成机理 (KDO 8-P）从阿拉伯糖5-磷酸（A5-P）和磷酸烯醇丙酮酸（PEP） 由酶KDO 8-P合酶（EC 4.1.2. 16），一种酶参与 所述脂质体的脂多糖区的脂质A部分的生物合成 革兰氏阴性菌的细胞被膜，2.形成机制 来自赤藓糖的3-脱氧-D-阿拉伯-庚酮糖酸7-磷酸（DAH 7-P） 4-磷酸（E4-P）和PEP由DAH 7-P合酶[EC 4.1.2.15]，催化第一个承诺步骤的酶， 生物合成芳香族氨基酸和各种芳香族二级 代谢物。具体的施舍侧重于使用不同的技术， “可视化”潜在的四面体中间体。这些方法包括 快速混合，脉冲流ESIMS技术，以确认形成一个 反应中间体和旋转回波双共振NMR实验 的低温底物包埋在酶中，以观察中间体。快速 将开发温度淬火方法，以隔离潜在的 用于NMR结构研究的中间体。多核核磁共振分析 酶与各种标记的底物类似物的相互作用将被 用于观察失败的中间体和底物类似物， “稳定”这种潜在的失败中间体将用于进一步 了解这些反应的机制。金属离子的作用将 也被调查。基于X射线的定点诱变研究 晶体学数据，将被利用，以进一步了解 酶功能对底物结合的贡献，单体界面 相互作用和酶的机制。

DESCRIPTION: (Provided by Applicant) Health care providers are challenged daily by an increasing resistance of pathogenic bacteria to their antibacterial arsenal. To overcome this problem, it is necessary to design new and innovative antibiotics with totally different modes of action so that, no cross-resistance with present agents 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 on the enzymes 3-deoxy-D-mannoo-octulosonate 8-phosphate and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The information will prove useful in the design of selective inhibitors of these unique enzymes, namely a new generation of mechanistically diverse antibiotics. The goals of this project are to establish 1. The mechanism for the formation of 3-deoxy-D-manno-octulosonic 8-phosphate (KDO 8-P) from arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) catalyzed by the enzyme KDO 8-P synthase (EC 4.1.2. 16), an enzyme involved in the biosynthesis of the lipid A portion of the lipopolysaccharide region of the cell envelope of gram-negative bacteria, 2. The mechanism for the formation 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) from erythrose 4-phosphate (E 4-P) and PEP catalyzed by the enzyme DAH 7-P synthase [EC 4.1.2.15], the enzyme that catalyzes the first committed step in the biosynthesis the aromatic amino acids and various aromatic secondary metabolites. The specific alms focus on the use of diverse techniques to "visualize" the potential tetrahedral intermediate. These methods include a rapid mixing, pulsed-flow ESIMS technique to confirm the formation of a reaction intermediate(s) and rotational-echo double-resonance NMR experiments of sub-zero substrate entrapped in enzyme to observe the intermediate. A rapid temperature quench methodology will be developed to isolate the potential intermediate(s) for NMR structural studies. Multinuclear NMR analysis of the interaction of the synthases with various labeled substrate analogues will be utilized to observe abortive intermediates and substrate analogs designed to "stabilize" this potential abortive intermediate(s) will be used to further understand the mechanisms of these reactions. The role of the metal ion will also be investigated. Site-directed mutagenesis studies, based on x-ray crystallographic data, will be exploited to gain further insight into the contribution of enzyme functionalities to substrate binding, monomer interface interactions and to the mechanism of the enzyme.