MECHANISTIC ENZYMOLOGY OF THIAMINE BIOSYNTHESIS

硫胺素是人类饮食的重要组成部分， 市售食品（RDA = 1.4 mg）。缺乏这种维生素会导致 一种叫做脚气的神经系统疾病在分子水平上，硫胺素 依赖酶在碳水化合物中起着特别重要的作用， 代谢，包括转酮醇酶，α-酮酸脱羧酶， α-酮酸氧化酶、乙酰乳酸合酶和乙醛酸合酶。 我们研究的长期目标是了解 硫胺素生物合成途径的酶学和调节 大肠埃希菌这些研究之所以重要，有三个原因。 首先，由于这种维生素是人类饮食的必需成分， 对了解它是如何生物合成的很重要。其次从 从基础科学的角度来看，生物合成途径涉及一个 异常大量的新型机械化学。最后，我们的研究 将有助于构建过表达菌株， 用于商业生产硫胺素或其组分（2，000 吨/年生产的全合成）。 在这项资助中，我们将进行一个完整的机械表征， 参与噻唑和嘧啶偶联的酶 硫胺素（硫胺素磷酸焦磷酸化酶）。同时，我们 将进行一系列的实验， 参与噻唑生物合成。一旦我们确定了 反应后，我们将对每种酶进行系统的机理研究。

Thiamin 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 a neurological disease called beri-beri. At the molecular level, thiamin dependent enzymes play a particularly important role in carbohydrate metabolism and include transketolases, alpha-ketoacid decarboxylases, alpha-ketoacid oxidases, acetolactate synthase and glyoxalate synthase. The long range goals of our research are to understand the mechanistic enzymology and regulation of the thiamin biosynthetic pathway in Escherichia coli. These studies are significant for three reasons. Firstly, since this vitamin is a required component of the human diet, it is important to understand how it is biosynthesized. Secondly, from the perspective of basic science, the biosynthetic pathway involves an unusually large amount of novel mechanistic chemistry. Lastly, our studies will facilitate the construction of overexpression strains that may be of use for the commercial production of thiamin or its components (2,000 tons/year produced by total synthesis). In this grant, we will carry out a complete mechanistic characterization of the enzyme involved in the coupling of the thiazole and the pyrimidine moieties of thiamin (thiamin phosphate pyrophosphorylase). In parallel, we will carry out a series of experiments designed to identify the reactions involved in the thiazole biosynthesis. Once we have identified specific reactions, we will initiate systematic mechanistic studies on each enzyme.