Mechanistic Studies of Flavin Dependent Thymidylate Synthase

黄素依赖性胸苷酸合成酶的机理研究

• 批准号: 8787590
• 负责人: AMNON KOHEN
• 金额: $ 29.07万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 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/8787590
• 关键词: Acids; Anabolism; Anthrax disease; Antibiotics; Archaea; Bacteria; Binding; Biochemistry; Biological; Botulism; Catalysis; Chemicals; Chemistry; Code; Commit; Complex; DNA; DNA biosynthesis; Development; Dihydrofolate Reductase; Drug Design; Drug Targeting; Drug resistance; Enzyme Kinetics; Enzymes; Eukaryota; FADH2; Flavins; Folate; Genes; Genetic; Goals; Government Agencies; 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; Solutions; 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; public health relevance; research study; structural biology; thymidylate synthase-dihydrofolate reductase; tool

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(申请人提供)：胸腺嘧啶(DNA碱基)的生物合成在所有生物体中都是必不可少的。在人类和其他真核生物中，这种生物合成的最后一步是由Thya/Tyms编码的胸苷合成酶(TSase)催化的，其辅助因子由FOLA编码的二氢叶酸还原酶(DHFR)循环。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中，胸腺X编码的黄素依赖胸苷合成酶(FDTS)为胸腺嘧啶提供了一种替代的生物合成途径。乍一看，FDTS似乎只是结合了TSase和DHFR的活性；它具有与双功能TSase-DHFR相同的反应物和产物。然而，FDTS具有与人类对应的非常不同的遗传、结构和机械特性。FDTS的催化机制尚不清楚；它们没有已知的有效抑制剂；经典TSase或DHFRs的抑制剂不能有效地抑制FDTS。如果了解它们的作用机制，合理的抑制剂设计可能会导致新的低毒抗生素类药物的出现。本建议旨在研究FDTS催化的化学机理。这项研究引起了更广泛的兴趣，因为初步研究表明，FDTS的化学机制不同于双功能TSase-DHFRs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、替代辅助因子、X射线结晶学以及假定的中间体的合成和测试。目前这些不同的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：诱捕和识别中间体。具体目标2：检查假定的外环亚甲基中间体。具体目标3：结构研究。具体目标4：使用5-去氮杂-FADH2作为机械工具。