Mechanism of cofactor biosynthesis required for chronic bacterial infection

慢性细菌感染所需辅因子生物合成机制

• 批准号: 9102114
• 负责人: Kenichi Yokoyama
• 金额: $ 30.46万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9102114
• 关键词: Active Sites; Anabolism; Animal Mammary Glands; Antibiotics; Antitubercular Agents; Bacteria; Bacterial Infections; Biological Assay; Carbon; Characteristics; Chronic; Communicable Diseases; Complex; Coupling; Data; Development; Environment; Enzymes; Felis catus; Free Radicals; Future; Goals; Growth; Guanine; Guanosine; Health; Hypoxia; In Vitro; Individual; Infection; Isotope Labeling; Knowledge; Lead; Ligands; Lytic; Methods; Modeling; Molecular Conformation; Movement; Mus; Mycobacterium tuberculosis; N-terminal; Nitrogen; Nutrient; Oxidation-Reduction; Pathway interactions; Proteins; Pseudomonas aeruginosa; Public Health; Reaction; Recurrence; Reporting; Research; Resistance; Ribose; Role; Series; Structure; Structure-Activity Relationship; Testing; Variant; Vertebral column; X-Ray Crystallography; acute symptom; analog; base; cofactor; combat; design; enzyme activity; enzyme biosynthesis; in vivo; inhibitor/antagonist; inorganic phosphate; insight; molybdenum cofactor; mutant; novel therapeutics; pathogenic bacteria; pyranopterin; tripolyphosphate

 DESCRIPTION (provided by applicant): Molybdenum cofactor (Moco) is a redox cofactor essential for bacterial growth under hypoxic and nutrient limiting environments, and therefore, is essential for persistence of pathogenic bacteria in mammalian hosts. Chronic bacterial infections are resistant to many antibiotics and cause the recurrence of acute symptoms. Although Moco biosynthesis has been shown to be essential for some pathogenic bacteria to cause chronic infections, the development of specific inhibitors has been hampered by a lack of understanding of the functions and mechanisms of the biosynthetic enzymes. The long-term goal of this project is to provide enzymological understanding of Moco biosynthesis in bacteria and its role in infectious disease. The current application focuses on the first committed step(s) of Moco biosynthesis where the characteristic pyranopterin structure of Moco is synthesized from guanine 5'-triphosphate (GTP). This transformation proceeds through an unprecedented mechanism in which the C-8 of GTP is inserted between the ribose C2' and C3'. While two enzymes (MoaA and MoaC) are known to be responsible for this transformation, their individual functions are currently under active debate. Recently, we reported the isolation of 3',8-cyclo- dihydro-GTP (3',8-cH2GTP) from in vitro MoaA assay solutions, and proposed that MoaA catalyzes the conversion of GTP to 3',8-cH2GTP, while MoaC catalyzes the conversion of 3',8-cH2GTP to cyclic pyranopterin monophosphate. In this application, we will test this hypothesis though three Specific Aims. In Aim 1, the catalytic function and mechanism of MoaA will be investigated using the purification/derivatization-free 13C NMR method and substrate analogs. In Aim 2, the structure-function relationship of MoaC will be investigated based on X-ray crystallography and in vivo and in vitro enzyme activity assays. In Aim 3, putative MoaC reaction intermediates will be captured using MoaC active-site mutants or substrate analogs. The proposed research is significant because it will provide mechanistic insights into Moco backbone formation as well as the scientific basis for the future development of Moco biosynthesis inhibitors.

 描述（由申请人提供）：钼辅因子（Moco）是在缺氧和营养限制环境下细菌生长所必需的氧化还原辅因子，因此，对于哺乳动物宿主中病原菌的持久性至关重要。慢性细菌感染对许多抗生素具有耐药性，并导致急性症状复发。尽管已经证明Moco生物合成对于一些致病菌引起慢性感染是必不可少的，但是由于缺乏对生物合成酶的功能和机制的了解，特异性抑制剂的开发受到阻碍。该项目的长期目标是提供对细菌中Moco生物合成及其在感染性疾病中的作用的酶学理解。本申请集中于Moco生物合成的第一个关键步骤，其中Moco的特征性吡喃蝶呤结构由鸟嘌呤5 '-三磷酸（GTP）合成。这种转化通过一种前所未有的机制进行，其中GTP的C-8插入核糖C2'和C3'之间。虽然已知两种酶（MoaA和MoaC）负责这种转化，但它们各自的功能目前正在积极辩论中。最近，我们报道了从体外MoaA测定溶液中分离3 '，8-cyclo- dihydro-GTP（3'，8-cH_2GTP），并提出MoaA催化GTP转化为3 '，8-cH_2GTP，而MoaC催化3'，8-cH_2GTP转化为环吡喃蝶呤一磷酸。在本申请中，我们将通过三个具体目标来检验这一假设。在目标1中，MoaA的催化功能和机制将使用纯化/衍生化自由13 C NMR方法和底物类似物进行研究。目标2：基于X射线晶体学和体内、体外酶活性测定，研究MoaC的结构与功能的关系。在目标3中，将使用MoaC活性位点突变体或底物类似物捕获推定的MoaC反应中间体。这项研究具有重要意义，因为它将为Moco骨架的形成提供机理上的见解，并为未来开发Moco生物合成抑制剂提供科学依据。