MECHANISTIC ENZYMOLOGY OF THIAMIN BIOSYNTHESIS

硫胺素是人类饮食的重要组成部分，并添加到许多商业食品（RDA = 1.4毫克）。 缺乏这种维生素会导致一种叫做脚气病的神经系统疾病。 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，包括转酮醇酶、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从机理和结构的角度完整地理解大肠杆菌中硫胺素生物合成的机理酶学。 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，因此了解它是如何生物合成的非常重要。 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新型机械化学。最后，我们的研究将有助于构建可用于商业生产硫胺素或其组分（通过全合成生产2，000吨/年）的过表达菌株。在此资助期内，我们将完成对硫胺素磷酸合酶和硫胺素酶I的机制研究。 同时，我们将进行一系列实验，旨在确定涉及从半胱氨酸到66个氨基酸载体肽的酰基腺苷酸的硫转移的反应。 还将使用过表达的ThiFSGH和ThiI研究从酪氨酸、1-脱氧-D-木酮糖-5-磷酸重建噻唑组装体。 一旦我们确定了硫转移和噻唑形成中的特定反应，我们将开始对所涉及的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 and acetolactate synthase. The long range goals of our research is the complete understanding of the mechanistic enzymology of thiamin biosynthesis in Escherichia coli from a mechanistic and structural perspective. 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 period, we will complete our mechanistic studies on thiamin phosphate synthase and on thiaminase I. In parallel, we will carry out a series of experiments designed to identify the reactions involved the sulfur transfer from cysteine to the acyl adenylate of a 66 amino acid carrier peptide. The reconstitution of the thiazole assembly from tyrosine, 1-deoxy-D-xylulose-5-phosphate will also be investigated using overexpressed ThiFSGH and ThiI. Once we have identified specific reactions in the sulfur transfer and in the thiazole formation, we will initiate systematic mechanistic studies on each of the enzymes involved.