Understanding trehalose synthesis and utilization in mycobacteria

了解分枝杆菌中海藻糖的合成和利用

• 批准号: 8596082
• 负责人: Donald R Ronning
• 金额: $ 34.66万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-20 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8596082
• 关键词: Active Sites; Affect; Anabolism; Antigens; Back; Binding; Biological; Carbohydrates; Catalysis; Cell Wall; Cells; Chemicals; Communicable Diseases; Communities; Complex; Cord Factors; Cytoplasm; Development; Disaccharides; Drug Targeting; Drug resistance; Drug usage; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Genetic; Genome; Genus Mycobacterium; Glucans; Goals; In Vitro; Kinetics; Knowledge; MALDI-TOF Mass Spectrometry; Maltose; Membrane; Metabolic Pathway; Metabolism; Modification; Molecular; Mutation; Mycobacterium tuberculosis; Mycolic Acid; Nature; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Physiology; Play; Polysaccharides; Positioning Attribute; Process; Production; Protein Dephosphorylation; Proteins; Recycling; Research; Research Project Grants; Roentgen Rays; Role; Staging; Structure; Structure-Activity Relationship; Testing; Therapeutic; Trehalose; Tuberculosis; Virulence; World Health Organization; analog; arabinogalactan; base; design; drug development; ebselen; enzyme mechanism; in vitro testing; in vivo; inhibitor/antagonist; inorganic phosphate; insight; interdisciplinary approach; killings; mutant; mycobacterial; mycolate; public health relevance; research study; sugar; tool; tuberculosis drugs

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.

描述（由申请人提供）：该提案的总体目标是更好地了解分枝杆菌中海藻糖的生物合成、利用和回收的关键步骤。 二糖海藻糖对于分枝杆菌生理学的多个方面都很重要，并且已被证明对于生存力至关重要，导致海藻糖单分枝菌酯生产和出口的下游利用途径也是如此。用于构建外膜的海藻糖的回收对于结核分枝杆菌毒力很重要，并且用于延长胞质葡聚糖。 该研究项目拥有 3 个独立的方面，均与海藻糖代谢相关。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，作为了解其在合成分枝杆菌葡聚糖中的功能的基础。由于 GlgE 的抑制可促进结核分枝杆菌的快速杀死，因此将合成基于机制的抑制剂并与稳态动力学结合使用，以更好地了解酶机制。将进行 X 射线晶体学研究来表征与 GlgE 底物的相互作用，这将构成针对 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依布硒啉杀灭分枝杆菌的机制。我们已经证明，依布硒啉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。体内和离体实验将鉴定细菌培养物中哪些分枝杆菌蛋白被依布硒啉修饰。 第三个目标将表征海藻糖生物合成途径最后一步的酶（海藻糖磷酸磷酸酶 2）的结构-功能关系。稳态动力学将用于研究活性位点突变和体外抑制的影响。无活性突变体也将用于平衡结合研究，以更好地了解底物选择性。该信息将为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增进我们对海藻糖代谢途径的了解。用于形成菌膜的结构单元、分枝酸和海藻糖单分枝酸酯的生物合成是一线和二线抗结核药物的已知靶标。因此，期望进一步明确生物合成 导致海藻糖单分枝菌酯的途径和表征将分枝菌酸附着到菌膜上的酶将为抗结核药物的开发提供新的见解。预计这项研究将扩展可用的结核分枝杆菌药物靶点，将海藻糖生物合成和利用途径中的酶包括在内。