Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

• 批准号: 7322118
• 负责人: STEVEN E EALICK
• 金额: $ 27.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7322118
• 关键词: Anabolism; Bacillus subtilis; Beriberi; Binding; Biochemical; Branched-Chain Amino Acids; Chemicals; Chemistry; Complement; Complex; Cysteine; D-xylulose-5-phosphate; DNA Sequence Rearrangement; Diet; Encephalopathies; Enzymatic Biochemistry; Enzymes; Facility Construction Funding Category; Fermentation; Flavoring; Food Additives; Glycine; Goals; Human; Laboratories; Life; Membrane; Metabolism; Numbers; Organism; Paper; Pathway interactions; Play; Production; Protein Binding; Protein Overexpression; Proteins; Pyrimidine; Pyrimidines; Reaction; Recombinant Proteins; Regulation; Research; Role; Structure; System; Thiamin Pyrophosphokinase; Thiamin-phosphate kinase; Thiamine; Thiamine Pyrophosphate; Thiazoles; Time; Universities; Vitamins; X-Ray Crystallography; Yeasts; analog; carbanion; carbohydrate metabolism; cofactor; deprivation; enzyme substrate; mutant; periplasm; protein protein interaction; thiamin phosphate synthase; thiaminase II; thiamine-binding protein; uptake

DESCRIPTION (provided by applicant): Thiamin is an essential cofactor in all living systems and is a required component of the human diet. Short term deprivation results in beri beri and Wemicke's encephalopathy and prolonged deprivation is lethal. Thiamin is also an important commercial chemical; it is widely used as a food additive and as a flavoring agent and annual production is on the order of 3,300 tons. Thiamin pyrophosphate, the active form of vitamin B1, plays an important role in carbohydrate metabolism and in branched-chain amino acid metabolism where it stabilizes acyl carbanion intermediates. Thiamin biosynthesisis not yet well understood and the reconstitutionof the pyrimidine and the thiazole moieties has only recently been accomplished in a defined biochemical system. In B. subtilis, thiamin pyrophosphate is synthesized from glycine, deoxy-D-xylulose 5-phosphate, cysteine and aminoimidizaole ribotide. The biosynthetic pathway is complex and uses 14 gene products. We have previously determined the structures of five thiamin biosynthetic enzymes and used these structures to support mechanistic studies. Our proposal has four specific aims. In aim 1, we will study the formation of the thiazole moiety by determining the structures of ThiF and ThiG, as well as the structures of the stable ThiSG and ThiFS complexes. The second specific aim describes structural studies on ThiC, the enzyme required for the formation of the pyrimidine moiety. In aim 3, we will study thiamin regulation and uptake by determining the structures of Tenl and TBP, respectively. The studies described in the first three specific aims will complete the structural characterization of the major bacterial thiamin biosynthetic pathway (excepting the membrane bound transport system). In the final specific aim, we will begin studies on thiamin biosynthesis in yeast, which proceeds by a very different pathway, by determining the structures of Thi4 and Thi5 - the only identified proteins involved in the biosynthesis of the thiazole and pyrimidine moieties in this organism. For all enzymes, we will also determine the structures of complexes and mutants, as needed, to understand the catalytic mechanisms. These studies will result in (1) an understanding of the biosynthesis of a vitamin required for all forms of life, (2) a mechanistic understanding of the unprecedented chemistry used for thiamin biosynthesis and (3) approaches for the construction of overexpression strains that can be used for the commercial production of thiamin by fermentation.

描述（由申请人提供）：硫胺素是所有生命系统中必需的辅助因子，也是人类饮食的必需成分。短期剥夺会导致脚气病和韦米克脑病，长期剥夺则致命。硫胺素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，年产量约为3300吨。焦磷酸硫胺素是维生素 B1 的活性形式，在碳水化合物代谢和支链氨基酸代谢中发挥着重要作用，可稳定酰基碳负离子中间体。硫胺素的生物合成尚未得到充分了解，嘧啶和噻唑部分的重建最近才在确定的生化系统中完成。在枯草芽孢杆菌中，焦磷酸硫胺素由甘氨酸、脱氧-D-木酮糖 5-磷酸、半胱氨酸和氨基咪唑核苷合成。生物合成途径很复杂，使用 14 种基因产物。我们之前已经确定了五种硫胺素生物合成酶的结构，并使用这些结构来支持机理研究。我们的提案有四个具体目标。在目标 1 中，我们将通过确定 ThiF 和 ThiG 的结构以及稳定的 ThiSG 和 ThiFS 复合物的结构来研究噻唑部分的形成。第二个具体目标描述了 ThiC（形成嘧啶部分所需的酶）的结构研究。在目标 3 中，我们将通过分别确定 Tenl 和 TBP 的结构来研究硫胺素的调节和吸收。前三个具体目标中描述的研究将完成主要细菌硫胺素生物合成途径（膜结合转运系统除外）的结构表征。在最终的具体目标中，我们将开始研究酵母中的硫胺素生物合成，该生物合成通过一种非常不同的途径进行，通过确定 Thi4 和 Thi5 的结构，这是唯一已识别的参与该生物体中噻唑和嘧啶部分生物合成的蛋白质。对于所有酶，我们还将根据需要确定复合物和突变体的结构，以了解催化机制。这些研究将导致（1）了解所有生命形式所需的维生素的生物合成，（2）对用于硫胺素生物合成的前所未有的化学的机械理解，以及（3）构建过度表达菌株的方法，该菌株可以用于发酵生产硫胺素的商业化生产。