The mechanistic enzymology of thiamin biosynthesis

硫胺素生物合成的机械酶学

• 批准号: 6630846
• 负责人: TADHG P. BEGLEY
• 金额: $ 35.57万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6630846
• 关键词: Bacillus subtilis; Escherichia coli; active sites; alkyltransferase; chemical kinetics; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; eukaryote; ionic bond; microorganism metabolism; prokaryote; proteomics; site directed mutagenesis; thiamine; thiamine pyrophosphate; thiazoles; vitamin biosynthesis; yeasts

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.

描述（由申请人提供）：硫胺素是人类饮食的重要组成部分，并被添加到许多商业食品（RDA = 1.4 mg）中。这种维生素缺乏会导致神经系统疾病贝里伯利和沃尼克脑病。在分子水平上，硫胺素依赖性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​​酸合成酶。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全机械理解。我们的研究很重要，原因有三个。首先，由于这种维生素是人类饮食的必需组成部分，也是各种生活形式的必需辅助因子，因此了解其生物合成是如何化为生物合成非常重要的。其次，从基础科学的角度来看，生物合成途径涉及异常大量的前所未有的化学：复杂的氧化凝结反应的机制，使得噻唑和空气转化为嘧啶的转化涉及的显着重新安排尚未阐明。最后，我们的研究将促进过表达菌株的构建，这些菌株将用于硫胺素或其成分的商业生产（总合成产生的3500吨/年）。在这一赠款期间，我们将对噻唑和嘧啶形成反应进行机械研究，启动对酵母中硫胺素生物合成的研究，筛选肽库，以抑制硫胺素生物合成的抑制剂，并评估将硫胺素用作精选蛋白质组探针的使用。