Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

• 批准号: 7152950
• 负责人: STEVEN E EALICK
• 金额: $ 27.9万
• 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/7152950
• 关键词: Structural; Studies; Enzymes; Thiamin; Biosynthesis

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不可少的辅助因子，也是人类饮食的必需成分。短期的剥夺导致脚气病和韦米克脑病，长期的剥夺是致命的。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3，300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起着重要作用，在支链氨基酸代谢中，焦磷酸硫胺素稳定酰基碳负离子中间体。硫胺素的生物合成还没有被很好地理解，嘧啶和噻唑部分的重组直到最近才在一个确定的生物化学系统中完成。在B。在枯草芽孢杆菌中，硫胺素焦磷酸是由甘氨酸、脱氧-D-木酮糖5-磷酸、半胱氨酸和氨基咪唑核苷酸合成的。生物合成途径很复杂，使用14种基因产物。我们以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制研究。我们的建议有四个具体目标。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对ThiC的结构研究，该酶是形成嘧啶部分所需的。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摄取。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的结构表征（膜结合转运系统除外）。在最后的具体目标，我们将开始研究硫胺素在酵母中的生物合成，这是通过一个非常不同的途径，通过确定的结构的Thi4和Thi5-唯一确定的蛋白质参与生物合成的噻唑和嘧啶部分在这个有机体。对于所有的酶，我们也将确定复合物和突变体的结构，根据需要，以了解催化机制。这些研究将导致（1）对所有生命形式所需的维生素的生物合成的理解，（2）对用于硫胺素生物合成的前所未有的化学的机械理解和（3）用于构建可用于通过发酵商业生产硫胺素的过表达菌株的方法。