Mechanistic structure-function studies of the type II citrate synthase of gram-negative bacteria

革兰氏阴性菌与植物、动物和人类的多种不同疾病有关，具有严重的经济和健康后果。这些问题的范围从农作物的软腐病到加工食品的污染，包括许多最危险的人类病原体（大肠杆菌、淋病奈瑟菌、铜绿假单胞菌、伤寒沙门氏菌、霍乱弧菌等）。同样值得注意的是，革兰氏阴性细菌有一种独特的能量产生酶，柠檬酸合成酶，被称为II型，而大多数其他生物，包括人类，表达这种酶的I型二聚体版本。II型柠檬酸合成酶形成六聚体，通过NADH的强变构抑制进行代谢调节。因此，这些酶表现出T（无活性，结合NADH）和R（活性，结合底物）状态之间的构象平衡。在本应用程序中概述的研究计划中，提出了一系列强协同作用的机制和抑制剂发现实验。目标不仅是全面了解II型柠檬酸合酶独特的变构功能，而且还探索利用这一知识开发新型抗菌药物的可能性，将这些酶锁定在T失活状态，从而通过阻断革兰氏阴性菌的能量供应实现致病性清除。远程变体控制作为抗菌控制的范例是一种全新的根除病原体的方法，这一过程传统上依赖于直接活性位点抑制。我们最近在一项小型试点筛选研究中证明了变弹性锁定方法的有效性，该研究确定了三种新的潜在抗菌药物。在此基础上，我们建议对II型柠檬酸合酶进行以下机制研究：从结构上表征r态；绘制变构信号传递路径；表征变构导向活性位点展开的性质。为了进一步实现抗菌抑制剂发现的目标，我们进一步建议：扩大我们成功的试点筛选计划；功能测试确定了潜在的抑制剂；并对观察到的变构锁抑制模式进行了结构表征。

Gram-negative bacteria are involved in a wide range of different diseases in plants, animals and humans, with severe economic and health consequences. These range from soft rot in agricultural crops, to contamination of processed foods, and include many of the most dangerous human pathogens (E. coli, N. gonorrhoea, P. aeruginosa, S. typhi, V. cholerae, etc.). Also notable is the fact that Gram-negative bacteria have a unique form of the energy producing enzyme, citrate synthase, designated Type II, whereas most other organisms, including humans, express the Type I dimeric version of this enzyme. Type II citrate synthases form hexamers that are metabolically regulated through strong allosteric inhibition by NADH. As such, these enzymes exhibit a conformational equilibrium between the T (inactive, binding NADH) and R (active, binding substrates) states. In the research program outlined in this application, a strongly synergistic series of mechanistic and inhibitor discovery experiments are proposed. The goal is to not only attain a comprehensive understanding of the unique allosteric function of Type II citrate synthases, but also to explore the possibility of using this knowledge to develop novel anti-bacterials that lock these enzymes into the T inactive state and thereby achieve pathogenic clearance by choking off the energy supply of Gram-negative bacteria. Remote allosteric control as a paradigm for anti-bacterial control is an entirely new approach to the eradication of pathogens, a process that has traditionally depended on direct active site inhibition. We have recently demonstrated the validity of the allosteric lock down approach in a small pilot screening study that identified three new potential anti-bacterials. To build on this success we propose the following mechanistic studies of the Type II citrate synthases: structurally characterizing the R-state; mapping the route of allosteric signal transmission; and, characterizing the nature of allosterically directed active site unfolding. To further the goals of anti-bacterial inhibitor discovery, we further propose to: expand our successful pilot screening program; functionally test identified potential inhibitors; and, structurally characterize the modes of allosteric lock inhibition observed.