Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

基本信息

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
项目类别：
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

项目摘要

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需的辅助因子，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。原核生物的大部分生物合成途径已广为人知。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。我们现在已经重建了嘧啶合酶的生物合成，并且刚刚开始揭示这种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回收途径，并开始探索硫胺素的运输。最后，在原核生物噻唑生物合成中首次发现的硫转移系统可能广泛存在。我们的提案有五个具体目标。在目标 1 中，我们将通过确定 ThiH、ThiG/ThiS 共价加合物以及 TenI 和 Thi4 与结合配体的复合物的结构，继续研究噻唑部分的形成。在目标 2 中，我们将通过确定 Thi5 和铁重组 ThiC 与结合配体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 Thi6 和 ThiE 的结构来研究硫胺素磷酸合酶。我们还将研究 ThiE 作为抗菌药物设计的目标。在目标 4 中，我们将通过确定 TenA 直向同源物、ThiY、YlmB、Thi20（TenA/ThiD 融合）和新鉴定的硫胺素酶 I 直向同源物的结构来研究硫胺素的回收和吸收。我们还将确定参与氧化硫胺素回收的酶的结构。在目标 5 中，我们将研究硫胺素硫转移系统的推广。我们将确定硫代喹菌素生物合成中相关蛋白质的结构以及从硫酸盐到硫代羧酸盐的新途径中蛋白质的结构。对于上述所有系统，我们将与 Tadhg Begley 合作来了解生物化学和机械酶学。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可用于通过发酵进行硫胺素的商业生产。公共健康相关性：本研究研究生物体合成、回收和运输硫胺素（维生素 B1）的生化途径。该研究对于利用发酵进行硫胺素的商业生产以及针对硫胺素生物合成的抗菌剂的开发具有重要意义。