Understanding trehalose synthesis and utilization in mycobacteria

描述（由申请人提供）：本提案的总体目标是更好地了解分枝杆菌中海藻糖生物合成、利用和回收的关键步骤。双糖海藻糖对分枝杆菌生理的多个方面都很重要，并且已被证明对生存能力至关重要，因为下游利用途径导致海藻糖单菌酸的生产和出口。用于构建外膜的海藻糖的再循环对结核分枝杆菌的毒力很重要，并用于延长胞质葡聚糖。本研究项目有三个独立的方面，都与海藻糖代谢有关。首先，我们将确定结核分枝杆菌GlgE促进底物结合和催化的结构特征，作为了解其在分枝杆菌葡聚糖合成中的功能的基础。由于GlgE的抑制促进了结核分枝杆菌的快速杀伤，基于机制的抑制剂将被合成并与稳态动力学结合使用，以更好地了解酶的机制。将进行x射线晶体学研究，以表征与GlgE底物的相互作用，这将形成针对GlgE的药物开发的基础。本研究的第二部分旨在进一步表征抗结核药物埃布selen杀死分枝杆菌的机制。我们已经证明ebselen通过共价修饰破坏酶活性位点并使其失活，从而强烈抑制抗原85C。在体内和离体进行的实验将确定哪些分枝杆菌蛋白在细菌培养中被埃布selen修饰。第三个目标将表征酶的结构-功能关系催化在从头海藻糖生物合成途径的最后一步，海藻糖磷酸磷酸酶2。稳态动力学将用于研究活性位点突变和体外抑制的影响。失活突变体也将用于平衡结合研究，以更好地了解底物选择性。这些信息将为在体内进行的研究提供信息，并确定TPP2是否是有效的药物靶点。这些研究的结果将用于提高我们对海藻糖代谢途径的认识。用于形成菌膜的构建块的生物合成，霉菌酸和海藻糖单菌酸，是已知的一线和二线抗结核药物的靶点。因此，期望进一步界定生物合成

DESCRIPTION (provided by applicant): The overarching goal of this proposal is to better understand key steps in the biosynthesis, utilization and recycling of trehalose in mycobacteria. The disaccharide trehalose is important for multiple aspects of mycobacterial physiology and has been shown to be essential for viability as is the downstream utilization pathway that leads to trehalose monomycolate production and export. Recycling of the trehalose used to build the outer membrane is important for M. tuberculosis virulence and is used to elongate a cytosolic glucan. This research project possesses 3 separate aspects that are all related to trehalose metabolism. First, we will identify structural features of M. tuberculosis GlgE that promote substrate binding and catalysis as a basis for understanding its function in synthesizing the mycobacteria glucan. Since inhibition of GlgE promotes rapid killing of M. tuberculosis, mechanism based inhibitors will be synthesized and used in conjunction with steady-state kinetics to better understand the enzyme mechanism. X-ray crystallographic studies will be performed to characterize interactions with GlgE substrates, which will form the basis for drug-development targeting GlgE. The second portion of this study aims to further characterize the mechanism of mycobacterial killing by the anti-tubercular drug ebselen. We have shown that ebselen strongly inhibits Antigen 85C through a covalent modification that disrupts the enzyme active site and inactivates it. Experiments performed in vivo and ex vivo will identify which mycobacterial proteins are modified by ebselen in a bacterial culture. The third aim will characterize the structure-function relationship of the enzyme catalyzing the final step in the de novo trehalose biosynthetic pathway, trehalose phosphate phosphatase 2. Steady-state kinetics will be used to study the effects of active site mutations and inhibition in vitro. Inactive mutant will also be used in equilibrium binding studies to better understand substrate selectivity. This information will inform studies performed in vivo and determine if TPP2 is a valid drug target. The results from these studies will be used to advance our knowledge of the metabolic pathways that use trehalose. The biosynthesis of the building blocks used to form the mycomembrane, mycolic acids and trehalose monomycolate, are known targets of first and second-line anti-tubercular drugs. Therefore, it is expected that further defining the biosynthetic pathway leading to trehalose monomycolate and characterizing the enzymes that attach mycolic acids to the mycomembrane will offer new insights for anti-tubercular drug development. It is expected that this study will extend the available Mycobacterium tuberculosis drug targets to include enzymes in the trehalose biosynthetic and utilization pathways.