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

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

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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）的生物合成在所有生物体中都是必不可少的。这种生物合成和其他真核生物的生物合成的最后一步是由Thya/Tyms编码的胸苷甲酯合酶（TSSASE）催化的，其辅助因子被fola编码的二氢酸盐还原酶（DHFR）循环回收。在几种人类病原体中，例如引起炭疽病，结核病，斑疹伤寒等的病原体，thyx编码的黄素依赖性胸苷合酶（FDTS）为胸腺素提供了替代的生物合成途径。乍一看，FDT似乎仅仅是结合了TSase和DHFR的活动。它具有与双功能的TSASE-DHFR相同的反应物和产物。但是，FDT与人类对应物具有巨大的遗传，结构和机械性能。 FDTS的催化机制尚不清楚。他们没有已知的有效抑制剂；经典TSases或DHFR的抑制剂不会有效抑制FDTS。如果已知的机制，理性抑制剂设计可能会导致新的抗生素药物，具有低毒性。该建议旨在研究FDTS催化的化学机制。这项研究具有更广泛的兴趣，因为初步研究表明，FDTS化学机制与双功能TSase-dhfr或任何其他已知的核苷酸甲基化机制不同。拟议的研究将采用广泛的方法论，包括同位素标记，反应中间人的单转捕集，稳态前和稳态酶动力学，时间分辨ESI-MS，诱变，替代辅助因子，替代辅助因子，X射线晶体学晶体学和测试的合成和假定Intermedises。这些不同的机械研究的发现将测试各种提出的机制，并将阐明该酶的神秘机制。提出了四个具体目标：具体目标1：中间体的陷阱和识别。具体目标2：检查推定的外环甲基中间体。特定目标3：结构研究。特定目标4：使用5-DEAZA-FADH2作为机械工具。