Mechanistic Studies of Flavin Dependent Thymidylate Synthase

描述（由申请人提供）：胸腺嘧啶（一种DNA碱基）的生物合成在所有生物体中都是必需的。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编码的胸苷酸合成酶（TSase）催化的，其辅因子由folA编码的二氢叶酸还原酶（DHFR）再循环。在几种人类病原体中，例如，那些引起炭疽、肺结核、斑疹伤寒和更多的疾病，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。初看起来，FDTS似乎只是将TSase和DHFR的活性联合收割机结合起来;它与双功能TSase-DHFR具有相同的反应物和产物。然而，FDTS与人类相比具有非常不同的遗传，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TS酶或DHFR的抑制剂不能有效抑制FDTS。如果它们的机制已知，合理的抑制剂设计可能导致具有低毒性潜力的新型抗生素药物。本研究旨在探讨FDTS催化的化学机理。这项研究具有更广泛的意义，因为初步研究表明FDTS的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。拟定的研究将采用广泛的方法，包括同位素标记、反应中间体的单次转换捕获、前稳态和稳态酶动力学、时间分辨ESI-MS、诱变、替代辅因子、X射线晶体学以及推定中间体的合成和检测。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。提出了四项具体目标：具体目标1：捕获和查明中间体。具体目标2：检查推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-脱氮-FADH 2作为机械工具。

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.