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

描述：(由申请人提供)医疗服务提供者每天都面临挑战 通过增加病原菌对其抗菌剂的抗药性 阿森纳。要克服这一问题，就必须设计出新的、创新的产品 抗生素具有完全不同的作用模式，因此，没有交叉耐药性 在目前的情况下，应该发生代理。大多数抗菌药物的作用方式是抑制 生物合成大分子所需的关键酶 微生物的生存能力。这种类型的方法的成功需要 在分子水平上对酶(S)有透彻的了解。的目标是 这项工作是为了收集有关酶的机制信息 3-脱氧-D-甘露-辛基-8-磷酸和3-脱氧-D-阿拉伯-七磺酸盐 7-磷酸合成酶。这些信息将被证明对设计 这些独特的酶的选择性抑制剂，即新一代 机械多样化的抗生素。该项目的目标是建立 1.3-脱氧-D-甘露-辛基-8-磷酸的形成机理 (KDO 8-P)由阿拉伯糖5-磷酸(A 5-P)和磷酸烯醇式丙酮酸(PEP)合成 由KDO 8-P合成酶(EC 4.1.2.16)，一种参与 猪瘟病毒脂多糖区脂类A的生物合成 革兰氏阴性细菌的细胞膜，2.形成机制 来自红血球的3-脱氧-D-阿拉伯-庚磺酸盐7-磷酸(DAH 7-P) DAH 7-P合成酶催化的4-磷酸(E4-P)和PEP 4.1.2.15]，该酶催化 生物合成芳香族氨基酸及各种芳香族次生物质 代谢物。具体的施舍侧重于使用不同的技术来 “想象”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 反应中间体(S)和旋转回波双共振核磁共振实验 将负温底物包埋在酶中观察中间体。一次快速的 将开发温度猝灭方法来隔离潜在的 核磁共振结构研究中间体(S)。多核核磁共振波谱分析 合成酶与各种标记底物类似物的相互作用将是 用于观察流产的中间体和底物类似物 稳定这一潜在的流产中间体(S)将被用来进一步 了解这些反应的机制。金属离子的作用将 也被调查。基于X射线的定点突变研究 结晶学数据，将被用来进一步洞察 酶官能度对底物结合、单体界面的贡献 相互作用和酶的作用机制。

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.