The mechanistic enzymology of thiamin biosynthesis

说明(申请人提供)：硫胺素是人类饮食中的一种基本成分，RDA为1.2毫克。它也是一种重要的商业化学品，广泛用作食品添加剂和调味剂。化学合成法的年产量约为3300吨。硫胺素依赖的酶在碳水化合物和支链氨基酸代谢中起着重要作用。在这个时候，我们对这类酶的机制理解是复杂的。相比之下，尽管硫胺素是第一个被发现的维生素，但我们对其生物合成的了解仍然不完整，因为生物合成途径复杂，涉及前所未有的反应化学。硫胺素由一种与嘧啶相连的噻唑组成。细菌中的噻唑部分是由脱氧-D-木糖-5-磷酸、甘氨酸和66个氨基酸的蛋白质硫代羧酸盐通过复杂的氧化缩合形成的。细菌中的嘧啶是由5-氨基咪唑核苷酸通过复杂的重排反应形成的。然后，将嘧啶与噻唑偶联，得到硫胺素磷酸，最后进行磷酸化，得到生物活性形式的维生素B1。我们对细菌中的噻唑形成的机理的了解现在处于深入阶段，我们最近已经确定了真核生物中的一些主要的噻唑形成的特征。对嘧啶并噻唑偶联反应也有了较好的认识，并对嘧啶碳正离子中间体进行了结构和动力学表征。相比之下，我们对细菌和真核生物中硫胺素嘧啶形成过程中涉及的非凡化学物质的机械理解仍处于早期阶段。在下一个资助期，我们建议继续对噻唑生物合成酶的机理进行表征，评估与噻唑形成有关的新型硫转移化学的普遍性，并对嘧啶生物合成酶进行机理和结构研究。此外，我们还将对酸降解硫胺素回收的酶学进行探讨。我们研究的长期目标是全面了解原核生物和真核生物中硫胺素生物合成的酶学机制，并阐明硫胺素代谢与细胞生理的其他方面之间的联系。我们的研究之所以意义重大，有四个原因。首先，重要的是要了解硫胺素是如何生物合成的，因为这种维生素是人类饮食中的必需成分，也是所有形式生命的基本辅因子。其次，生物合成途径涉及大量史无前例的生物化学。第三，我们的研究将有助于构建可用于发酵生产硫胺素的高表达菌株。最后，硫胺素生物合成酶的抑制剂可能为结核分枝杆菌等缺乏硫胺素运输系统的细菌的抗生素设计提供一种选择性策略。与公共健康相关我们感兴趣的是维生素B1(硫胺素)是如何在生命系统中组装的。这是一个重要的问题，因为维生素B1是人类饮食中的必需成分，对所有形式的生命都是必不可少的。硫胺素被添加到许多食物中，我们的研究将促进其通过发酵进行商业化生产。此外，我们的研究在设计针对结核病的抗生素方面具有潜在的应用价值。

DESCRIPTION (provided by applicant): Thiamin is an essential component of the human diet with an RDA of 1.2 mg. It is also an important commercial chemical and it is widely used as a food additive and flavoring agent. Annual production, by chemical synthesis, is on the order of 3,300 tons. Thiamin-dependent enzymes play an important role in carbohydrate and branched-chain amino acid metabolism. At this time, our mechanistic understanding of this class of enzymes is sophisticated. In contrast, while thiamin was the first vitamin identified, our understanding of its biosynthesis is still incomplete because the biosynthetic pathway is complex and involves unprecedented reaction chemistry. Thiamin consists of a thiazole linked to a pyrimidine. The thiazole moiety in bacteria is formed from deoxy-D-xylulose-5-phosphate, glycine, and a 66 amino acid protein thiocarboxylate via a complex oxidative condensation. The pyrimidine in bacteria is formed from 5-aminoimidazole ribonucleotide via a complex rearrangement reaction. The pyrimidine is then coupled to the thiazole to give thiamin phosphate and a final phosphorylation gives the biologically active form of vitamin B1. Our mechanistic understanding of thiazole formation in bacteria is now at an advanced stage and we have recently established some of the main features of thiazole formation in eukaryotes. The pyrimidine thiazole coupling reaction is also well understood and the pyrimidine carbocation intermediate has been structurally and kinetically characterized. In contrast our mechanistic understanding of the remarkable chemistry involved in the formation of the thiamin pyrimidine in bacteria and in eukaryotes is still at an early stage. In the next funding period, we propose to continue our mechanistic characterization of the thiazole biosynthetic enzymes, evaluate the generality of the novel sulfur transfer chemistry involved in thiazole formation, and carry out mechanistic and structural studies on the pyrimidine biosynthetic enzymes. In addition, we will explore the enzymology of the salvage of acid-degraded thiamin. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamin biosynthesis in both prokaryotes and eukaryotes and the elucidation of connections between thiamin metabolism and other aspects of cellular physiology. Our studies are significant for four reasons. First, it is important to understand how thiamin is biosynthesized because this vitamin is a required component of the human diet and an essential cofactor for all forms of life. Second, the biosynthetic pathway involves an unusually large amount of unprecedented biological chemistry. Third, our studies will facilitate the construction of overexpression strains that will be of use for the commercial production of thiamin by fermentation. Finally, inhibitors of thiamin biosynthetic enzymes may provide a selective strategy for antibiotic design in bacteria such as Mycobacterium tuberculosis that lack a thiamin transport system. PUBLIC HEALTH RELEVANCE We are interested in how vitamin B1 (thiamin) is assembled in living systems. This is an important problem because vitamin B1 is a required component of the human diet and is essential for all forms of life. Thiamin is added to many foods and our studies will facilitate its commercial production by fermentation. In addition, our research has potential applications in the design of TB specific antibiotics.