Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

• 批准号: 8517682
• 负责人: STEVEN E EALICK
• 金额: $ 31.84万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517682
• 关键词: Anabolism; Area; Beriberi; Biochemical; Biochemical Pathway; Biochemistry; Bioinformatics; Branched-Chain Amino Acids; Carrier Proteins; Chemicals; Chemistry; Clostridium; Collaborations; Complex; Coupling; Cyclophilins; Cysteine; D-xylulose-5-phosphate; DNA Sequence Rearrangement; Development; Diet; Drug Design; Drug Targeting; Enzymatic Biochemistry; Enzymes; Eukaryota; Fermentation; Flavoring; Food Additives; Glycine; Goals; Histidine; Homologous Gene; Human; Hydrolase; Inorganic Sulfates; Iron; Joints; Learning; Life; Ligand Binding; Metabolism; Mycobacterium tuberculosis; N-terminal; Natural regeneration; Organism; Orthologous Gene; Oxythiamine; Pathway interactions; Phosphotransferases; Physiological; Plants; Play; Process; Production; Prokaryotic Cells; Protein Biosynthesis; Proteins; Publications; Pyridoxal; Pyrimidine; Research; Ribonucleotides; Role; Structure; Sulfur; System; Thiamine; Thiamine Pyrophosphate; Thiazoles; Tyrosine; Ubiquitin; Unspecified or Sulfate Ion Sulfates; Vitamins; Wernicke Encephalopathy; Yeasts; adduct; antimicrobial drug; carbanion; carbohydrate metabolism; cofactor; deprivation; design; enzyme structure; inorganic phosphate; molybdenum cofactor; novel; overexpression; professor; protein structure function; public health relevance; reconstitution; research study; suicide enzyme; thiamin phosphate synthase; thiaminase II; thiamine thiazole; 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 of thiamin results in beri beri and Wernicke'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. Much of the biosynthetic pathway for prokaryotes is well understood. 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 now reconstituted the biosynthesis of the pyrimidine synthase and are just beginning to unravel the mechanistic details of this complex arrangement. In yeast, thiamin pyrophosphate is synthesized from glycine, cysteine, NAD, PLP and histidine and requires four enzymes. We now have basic understanding of the thiazole synthase, but little is known about the pyrimidine synthase. We have also discovered a new thiamin salvage pathway and are beginning to explore thiamin transport. Finally, the sulfur transfer system first discovered in the biosynthesis of the thiazole in prokaryotes is likely to be widespread. Our proposal has five specific aims. In aim 1 we will continue to study the formation of the thiazole moiety by determining the structures of ThiH, the ThiG/ThiS covalent adduct and complexes of TenI and Thi4 with bound ligands. In aim 2 we will study the formation of the pyrimidine by determining the structures of Thi5 and of iron-reconstituted ThiC with bound ligands. In aim 3, we will study the thiamin phosphate synthase by determining the structures of Thi6 and ThiE complexed with the recently identified carboxylthiazole. We will also investigate ThiE as a target for the design of antimicrobial agents. In aim 4 we will study thiamin salvage and uptake by determining structures of TenA orthologs, ThiY, YlmB, Thi20 (TenA/ThiD fusion) and newly identified thiaminase I orthologs. We will also determine the structures of enzymes involved in oxythiamin salvage. In aim 5, we will investigate the generalization of the thiamin sulfur transfer system. We will determine the structures of related proteins in thioquinilobactin biosynthesis and of proteins in a novel pathway from sulfate to thiocarboxylate. For all the systems described above, we will collaborate with Tadhg Begley to understand the biochemistry and mechanistic enzymology. 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. PUBLIC HEALTH RELEVANCE: This research studies the biochemical pathways by which organisms synthesize, salvage and transport thiamin (vitamin B1). The research has implications for the commercial production of thiamin using fermentation, and for the development of antimicrobial agents that target thiamin biosynthesis.

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。短期缺乏硫胺素会导致脚气病和韦尼克脑病，长期缺乏硫胺素会致命。硫胺素也是一种重要的商业化学品;它被广泛用作食品添加剂和调味剂，年产量约为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。原核生物的大部分生物合成途径都已得到很好的理解。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。我们现在已经重建了嘧啶合酶的生物合成过程，并且刚刚开始解开这个复杂排列的机制细节。在酵母中，焦磷酸硫胺素由甘氨酸、半胱氨酸、NAD、PLP和组氨酸合成，需要四种酶。我们现在对噻唑合酶有了基本的了解，但对嘧啶合酶知之甚少。我们还发现了一种新的硫胺素补救途径，并开始探索硫胺素的运输。最后，在原核生物中噻唑的生物合成中首次发现的硫转移系统可能是广泛存在的。我们的建议有五个具体目标。在目标1中，我们将通过确定ThiH、ThiG/ThiS共价加合物以及TenI和Thi 4与结合配体的复合物的结构来继续研究噻唑部分的形成。在目标2中，我们将通过确定Thi 5和具有结合配体的铁重构ThiC的结构来研究嘧啶的形成。在目标3中，我们将通过确定与最近鉴定的羧基噻唑复合的Thi 6和ThiE的结构来研究硫胺素磷酸合酶。我们还将研究ThIE作为抗菌剂设计的目标。在目标4中，我们将通过确定TenA直系同源物、ThiY、YlmB、Thi 20（TenA/ThiD融合物）和新鉴定的硫胺素酶I直系同源物的结构来研究硫胺素补救和摄取。我们还将确定参与氧硫胺素抢救的酶的结构。在目标5中，我们将研究硫胺素硫转移系统的推广。我们将确定在thioquinilobactin生物合成的相关蛋白质的结构和蛋白质在一个新的途径，从硫酸硫代羧酸。对于上述所有系统，我们将与Tadhg Begley合作，以了解生物化学和机械酶学。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，以及（3）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。 公共卫生相关性：这项研究研究生物体合成，抢救和运输硫胺素（维生素B1）的生化途径。该研究对使用发酵进行硫胺素的商业生产以及开发针对硫胺素生物合成的抗菌剂具有影响。