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成分，RDA为1.2 mg。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。通过化学合成，年产量约为3，300吨。硫胺素依赖酶在碳水化合物和支链氨基酸代谢中起重要作用。目前，我们对这类酶的机械理解是复杂的。相比之下，虽然硫胺素是第一种被发现的维生素，但我们对其生物合成的理解仍然不完整，因为生物合成途径很复杂，涉及前所未有的化学反应。硫胺素由一个噻唑和一个嘧啶连接而成。细菌中的噻唑部分由脱氧-D-木酮糖-5-磷酸、甘氨酸和66个氨基酸的蛋白硫代羧酸盐通过复杂的氧化缩合形成。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。然后嘧啶与噻唑偶联得到磷酸硫胺素，最后磷酸化得到维生素B1的生物活性形式。我们对细菌中噻唑形成的机理的理解现在处于高级阶段，我们最近已经确定了真核生物中噻唑形成的一些主要特征。嘧啶噻唑偶联反应也被很好地理解，并且嘧啶碳阳离子中间体已被结构和动力学表征。相比之下，我们对细菌和真核生物中硫胺嘧啶形成过程中所涉及的显著化学机制的理解仍处于早期阶段。在下一个资助期内，我们建议继续我们的噻唑生物合成酶的机械特性，评估的一般性的新的硫转移化学参与噻唑的形成，并进行嘧啶生物合成酶的机械和结构研究。此外，我们将探讨补救酸降解硫胺素的酶学。我们的研究的长期目标是完整的机制了解硫胺素生物合成的酶学在原核生物和真核生物和硫胺素代谢和细胞生理学的其他方面之间的联系的阐明。我们的研究之所以重要，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的必需成分，也是所有生命形式的重要辅助因子。第二，生物合成途径涉及大量前所未有的生物化学。第三，我们的研究将有助于构建过表达菌株，这些菌株将用于通过发酵进行硫胺素的商业生产。最后，硫胺素生物合成酶的抑制剂可能为缺乏硫胺素转运系统的细菌（如结核分枝杆菌）的抗生素设计提供选择性策略。 我们对维生素B1（硫胺素）在生命系统中的组装方式感兴趣。这是一个重要的问题，因为维生素B1是人类饮食的必需成分，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品中，我们的研究将有助于通过发酵进行商业生产。此外，我们的研究在结核特异性抗生素的设计中具有潜在的应用。

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.