Mechanistic Studies of Flavin Dependent Thymidylate Synthase

黄素依赖性胸苷酸合成酶的机理研究

基本信息

批准号：
9111948
负责人：
Daniel M Quinn
金额：
$ 29.07万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9111948
关键词：
Acids Anabolism Anthrax disease Antibiotics Archaea Bacteria Binding Biochemistry Biological Botulism Catalysis Chemicals Chemistry Code Complex DNA DNA biosynthesis Development Dihydrofolate Reductase Drug Design Drug Targeting Drug resistance Enzyme Kinetics Enzymes Eukaryota FADH2 Flavins Folic Acid Genes Genetic Goals Government Agencies Health Human Hydrogen Isomerism Label Laboratories Lead Life Light Lyme Disease Methodology Methods Methylation Modeling Molecular Biology Mutagenesis NADP Niacinamide Nucleotides Organic Chemistry Organism Peptic Ulcer Periodontal Diseases Pharmaceutical Preparations Pharmacologic Substance Physical Chemistry Preparation Property Public Health Reaction Recycling Structure Syphilis TYMS gene Testing Thermotoga maritima Thymidine Thymidylate Synthase Thymine Time Toxic effect Tuberculosis Typhus Water X-Ray Crystallography analog aqueous base biodefense carbene catalyst cofactor design enzyme mechanism inhibitor/antagonist interest pathogen research study structural biology thymidylate synthase-dihydrofolate reductase tool

项目摘要

DESCRIPTION (provided by applicant): The biosynthesis of thymine (a DNA base) is essential in all organisms. The last step in this biosynthesis in humans and other eukaryotes is catalyzed by thyA/TYMS-encoded thymidylate synthase (TSase), and its cofactor is recycled by the folA-encoded dihydrofolate reductase (DHFR). In several human pathogens, e.g., those causing anthrax, tuberculosis, typhus, and more, the thyX-encoded flavin-dependent thymidylate synthase (FDTS) provides an alternative biosynthetic path to thymine. At first glance, FDTS seems merely to combine the activities of TSase and DHFR; it has same reactants and products as bi-functional TSase-DHFR. However, FDTS has very different genetic, structural, and mechanistic properties than its human counterparts. The catalytic mechanism of FDTSs is not understood; they have no known potent inhibitors; and inhibitors of classical TSases or DHFRs do not efficiently inhibit FDTSs. Were their mechanism known, rational inhibitor design could lead to new classes of antibiotic drugs with the potential for low toxicity. This proposal aims at studies of the chemical mechanism of FDTS catalysis. This study is of broader interest as preliminary studies suggested that FDTS chemical mechanism is different from that of either bifunctional TSase- DHFRs or any other known mechanism of nucleotide methylation. The proposed studies will employ a broad arsenal of methodologies, including isotopic labeling, single-turnover trapping of reaction intermediates, pre- steady-state and steady-state enzyme kinetics, time-resolved ESI-MS, mutagenesis, alternative cofactors, X-ray crystallography, and the synthesis and testing of putative intermediates. The findings from these diverse mechanistic studies present will test various proposed mechanisms and will illuminate the enigmatic mechanism of this enzyme. Four specific aims are proposed: Specific Aim 1: Trapping and Identification of Intermediates. Specific Aim 2: Examination of the putative exocyclic methylene intermediate. Specific Aim 3: Structural studies. Specific Aim 4: Using 5-deaza-FADH2 as a mechanistic tool.

描述（由申请人提供）：胸腺嘧啶（DNA 碱基）的生物合成对于所有生物体都是必需的。人类和其他真核生物生物合成的最后一步是由 thyA/TYMS 编码的胸苷酸合酶 (TSase) 催化，其辅因子由 folA 编码的二氢叶酸还原酶 (DHFR) 回收。在几种人类病原体中，例如引起炭疽、结核病、斑疹伤寒等的病原体，thyX 编码的黄素依赖性胸苷酸合酶 (FDTS) 提供了胸腺嘧啶的替代生物合成途径。乍一看，FDTS似乎只是结合了TSase和DHFR的活性；它与双功能TSase-DHFR具有相同的反应物和产物。然而，FDTS 与人类对应物具有非常不同的遗传、结构和机制特性。 FDTS 的催化机制尚不清楚；他们没有已知的有效抑制剂；经典 TSase 或 DHFR 的抑制剂不能有效抑制 FDTS。如果其机制已知，合理的抑制剂设计可能会产生具有低毒性潜力的新型抗生素药物。该提案旨在研究FDTS催化的化学机制。这项研究引起了更广泛的兴趣，因为初步研究表明 FDTS 化学机制不同于双功能 TSase-DHFR 或任何其他已知的核苷酸甲基化机制。拟议的研究将采用广泛的方法学，包括同位素标记、反应中间体的单周转捕获、稳态前和稳态酶动力学、时间分辨ESI-MS、诱变、替代辅因子、X射线晶体学，以及假定中间体的合成和测试。这些不同机制研究的结果将测试各种提出的机制，并将阐明这种酶的神秘机制。提出了四个具体目标：具体目标 1：中间体的捕获和鉴定。具体目标 2：检查假定的环外亚甲基中间体。具体目标 3：结构研究。具体目标 4：使用 5-deaza-FADH2 作为机械工具。