The mechanistic enzymology of thiamin biosynthesis

硫胺素生物合成的机械酶学

• 批准号: 8034976
• 负责人: TADHG P. BEGLEY
• 金额: $ 10.63万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8034976
• 关键词: Acids; Active Sites; Amino Acids; Anabolism; Antibiotics; Aromatase; Bacteria; Binding; Biochemistry; Branched-Chain Amino Acids; Carbohydrates; Cell physiology; Chemicals; Chemistry; Complex; Coupled; Coupling; D-xylulose-5-phosphate; DNA Sequence Rearrangement; Detection; Development; Diet; Enzymatic Biochemistry; Enzymes; Eukaryota; Fermentation; Flavoring; Food; Food Additives; Funding; Glycine; Goals; Human; Imines; Iron; Kinetics; Life; Link; Metabolism; Mycobacterium tuberculosis; Organism; Pathway interactions; Phosphorylation; Physical condensation; Play; Post-Translational Protein Processing; Production; Prokaryotic Cells; Property; Proteins; Proteomics; Pyrimidine; Pyrimidines; Reaction; Research; Ribonucleotides; Role; Staging; Structure; Sulfur; System; Thiamin Metabolism Pathway; Thiamine; Thiazoles; Time; Vitamins; analog; antibiotic design; base; chemical synthesis; cofactor; design; gene cloning; inhibitor/antagonist; inorganic phosphate; interest; novel; overexpression; public health relevance; reconstitution; thiamin phosphate synthase; thiamine thiazole

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.

描述（由申请人提供）：硫胺素是人类饮食的基本成分，RDA为1.2毫克。它也是一种重要的商业化学品，被广泛用作食品添加剂和调味剂。化学合成的年产量约为3300吨。硫胺素依赖性酶在碳水化合物和支链氨基酸代谢中起重要作用。目前，我们对这类酶的机理理解是复杂的。相比之下，虽然硫胺素是第一个被发现的维生素，但我们对其生物合成的理解仍然不完整，因为生物合成途径非常复杂，涉及前所未有的反应化学。硫胺素由一个与嘧啶相连的噻唑组成。细菌中的噻唑部分是由脱氧- d -木醛糖-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-氨基咪唑核糖核苷酸经过复杂的重排反应形成的。然后，嘧啶与噻唑偶联形成硫胺磷酸，最后的磷酸化形成具有生物活性的维生素B1。我们对细菌中噻唑形成的机理理解现在处于高级阶段，我们最近确定了真核生物中噻唑形成的一些主要特征。嘧啶-噻唑偶联反应也得到了很好的理解，嘧啶碳正离子中间体的结构和动力学特征也得到了证实。相比之下，我们对细菌和真核生物中硫胺嘧啶形成的重要化学过程的机理理解仍处于早期阶段。在下一个资助期内，我们计划继续对噻唑类生物合成酶进行机理表征，评估涉及噻唑形成的新型硫转移化学的普遍性，并对嘧啶类生物合成酶进行机理和结构研究。此外，我们将探讨酸降解硫胺素回收的酶学。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机制的完整理解，以及阐明硫胺素代谢与细胞生理其他方面之间的联系。我们的研究之所以重要，有四个原因。首先，了解硫胺素是如何生物合成的是很重要的，因为这种维生素是人类饮食中必需的组成部分，也是所有生命形式的必要辅助因子。其次，生物合成途径涉及异常大量的前所未有的生物化学。第三，我们的研究将有助于构建可用于通过发酵商业化生产硫胺素的过表达菌株。最后，硫胺素生物合成酶抑制剂可能为缺乏硫胺素运输系统的细菌（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）如何在生命系统中组装感兴趣。这是一个重要的问题，因为维生素B1是人类饮食中必需的成分，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，我们的研究将促进其通过发酵的商业化生产。此外，我们的研究在结核病特异性抗生素的设计中具有潜在的应用价值。