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

革兰氏阴性菌涉及植物、动物和人类的各种不同疾病，造成严重的经济和健康后果。范围从农作物软腐病到加工食品污染，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产生能量的酶--柠檬酸合成酶，被指定为II型，而包括人类在内的大多数其他生物都表达这种酶的I型二聚体。II型柠檬酸合成酶形成六聚体，通过NADH强烈的变构抑制来代谢调节。因此，这些酶在T(非活性结合NADH)和R(活性结合底物)状态之间表现出构象平衡。在本申请概述的研究计划中，提出了一系列具有很强协同性的机理和抑制剂发现实验。其目标不仅是全面了解II型柠檬酸合成酶的独特变构功能，而且探索利用这一知识开发新型抗菌药物的可能性，该药物可将这些酶锁定在T非活性状态，从而通过阻断革兰氏阴性细菌的能量供应来实现病原菌的清除。远程变构控制作为一种抗菌控制的范例，是根除病原体的一种全新的方法，这一过程传统上依赖于直接的活性部位抑制。我们最近在一项小型先导性筛选研究中证明了变构锁定方法的有效性，该研究确定了三种新的潜在抗菌药物。为了建立在这一成功的基础上，我们建议对II型柠檬酸合成酶进行以下机制研究：从结构上表征R-态；绘制变构信号传递的路线；以及表征变构定向活性部位展开的性质。为了促进抗菌抑制剂发现的目标，我们进一步建议：扩大我们成功的中试筛选计划；从功能上测试已确定的潜在抑制剂；以及从结构上表征观察到的变构锁定抑制模式。

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.