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：
8111080
负责人：
STEVEN E EALICK
金额：
$ 33.03万
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/8111080
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 Delivery Systems Drug Design 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.