The mechanistic enzymology of thiamin biosynthesis

描述（由申请人提供）：硫胺素是人类饮食的重要组成部分，被添加到许多商业食品中（RDA = 1.4毫克）。缺乏这种维生素会导致神经系统疾病脚气病和韦尼克脑病。在分子水平上，硫胺素依赖酶在碳水化合物代谢中起着特别重要的作用，包括转酮酶、α -酮酸脱羧酶、α -酮酸氧化酶和乙酰乳酸合酶。我们研究的长期目标是对原核生物和真核生物中硫胺素生物合成的酶学的完整机制理解。我们的研究意义重大，原因有三。首先，由于这种维生素是人类饮食中必需的组成部分，也是所有生命形式的必要辅助因子，因此了解它是如何生物合成的很重要。其次，从基础科学的角度来看，生物合成途径涉及了异常大量的前所未有的化学反应：复杂的氧化缩合反应产生噻唑的机制以及AIR转化为嘧啶的显著重排尚未阐明。最后，我们的研究将有助于构建用于硫胺素或其成分商业化生产的过表达菌株（全合成产量为3500吨/年）。在此资助期内，我们将开展噻唑和嘧啶形成反应的机理研究，启动酵母中硫胺素生物合成的研究，筛选硫胺素生物合成抑制剂的肽库，并评估硫胺素作为选择性蛋白质组探针的用途。

DESCRIPTION (provided by applicant): Thiamine is an essential component of the human diet and is added to many commercial foods (RDA = 1.4 mg). Deficiency in this vitamin results in the neurological diseases beriberi and Wernicke encephalopathy. At the molecular level, thiamine dependent enzymes play a particularly important role in carbohydrate metabolism and include transketolases, alpha-ketoacid decarboxylases, alpha-ketoacid oxidases and acetolactate synthase. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamine biosynthesis in both prokaryotes and eukaryotes. Our studies are significant for three reasons. First, since this vitamin is a required component of the human diet and an essential cofactor for all forms of life, it is important to understand how it is biosynthesized. Second, from the perspective of basic science, the biosynthetic pathway involves an unusually large amount of unprecedented chemistry: The mechanism of the complex oxidative condensation reaction to give the thiazole and the remarkable rearrangement involved in the conversion of AIR to the pyrimidine have not yet been elucidated. Finally, our studies will facilitate the construction of overexpression strains that will be of use for the commercial production of thiamine or its components (3,500 tons/year produced by total synthesis). In this grant period, we will carry out mechanistic studies on the thiazole and the pyrimidine forming reactions, initiate studies on thiamine biosynthesis in yeast, screen a peptide library for inhibitors of thiamine biosynthesis and evaluate the use of thiamine as a selective proteome probe.