Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

• 批准号: 9112993
• 负责人: STEVEN E EALICK
• 金额: $ 35.02万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9112993
• 关键词: 5' -deoxyadenosine; Active Sites; Address; Amino Acids; Anabolism; Antibiotics; Archaea; Area; Bacillus (bacterium); Bacillus subtilis; Bacteria; Binding; Binding Sites; Biochemical; Branched-Chain Amino Acids; Carbohydrates; Catabolism; Catalysis; Catalytic Domain; Collaborations; Complex; Confusion; Consumption; Cysteine; Development; Diet; Enzymatic Biochemistry; Enzymes; Eukaryota; Fatigue; Fermentation; Food; Glycine; Goals; Health; Histidine; Human; Iron; Joints; Keto Acids; Left; Life; Mental Depression; Metabolism; Metal Binding Site; Modification; Organism; Orthologous Gene; Pathway interactions; Physical condensation; Plants; Play; Process; Production; Prokaryotic Cells; Publications; Pyrimidine; Reaction; Reporting; Research; Ribonucleotides; Ribose; Role; Side; Source; Staging; Structure; Sulfides; Sulfur; Symptoms; Testing; Texas; Thiamine; Thiamine Deficiency; Thiamine Pyrophosphate; Thiazoles; Water; Yeasts; analog; dehydroalanine; design; fungus; inorganic phosphate; monomer; mutant; novel; plant fungi; research study; ribulose; suicide enzyme; thiaminase II; thiamine thiazole

DESCRIPTION (provided by applicant): Thiamin is essential to forms of life. Thiamin pyrophosphate plays a critical role in the combustion of carbohydrates and in the synthesis of branched-chain amino acids. Thiamin deficiency causes fatigue, confusion, depression, and irritability, and if left untreated, can be fatal. Thiamin is biosynthesized by most bacteria, yeas, fungi and plants. Humans must obtain thiamin from their diets. The overall goal of our research is to understand the detailed mechanistic enzymology of thiamin biosynthesis and metabolism. The experiments are designed to fill important gaps in our understanding of the thiamin pyrimidine synthase in bacteria and plants (ThiC), the thiamin thiazole synthase in yeast (THI4), the thiamin pyrimidine synthase in yeast (THI5), and thiamin degrading enzymes (thiaminases). ThiC is a novel radical SAM enzyme and catalyzes the complex rearrangement of aminoimidazole ribonucleotide to the thiamin pyrimidine. We have recently determined a structure of ThiC with its [4Fe-4S] cluster. Unlike canonical radical SAM enzymes the cluster binding domain is tethered to the catalytic domain, and inserts through domain swapping into the active site of a twofold related monomer. Curiously, in our initial structure the cluster is located 25 � from the active site, suggesting that translocation of the domain must take place prior to catalysis. Yeast THI4 catalyzes the condensation of NAD, glycine, and cysteine to form adenylated carboxythiazole. We have shown the THI4 is an iron-dependent suicide enzyme and that the source of the thiazole sulfur atom is a cysteine side chain from THI4 itself. The structur of THI4 identified key active site residues, but we have not yet identified the iron binding site. n archaea, the yeast THI4 ortholog was shown to catalyze the isomerization is ribose 1,5-bisphosphate to ribulose 1,5-bisphosphate. We have shown that the archaeal THI4 ortholog also catalyzes synthesis of the thiamin thiazole, but uses sulfide as the sulfur source. We do not yet know if this dual activity is present in other THI4's. Yeast THI5 catalyzes a remarkable condensation of PLP and histidine to form the thiamin pyrimidine. We have shown that THI5 is an iron-dependent suicide enzyme and that the histidine side chain comes from THI5 itself. The structure of THI5 identified key active site residues, but we have not yet identified the iron binding site. For ThiC, THI4 and THI5, we will determine structures with substrates, products and analogs, identify metal binding sites, and use mutant enzymes to trap intermediates, thus filling in key mechanistic details. Thiaminase-I and II's are thiamin degrading enzymes that paradoxically often cluster with thiamin biosynthetic enzymes. The Bacillus subtilis thiaminase-II was shown to participate in thiamin salvage. We therefore hypothesize that salvage of degraded thiamin may be the general role of thiaminase-II's and that diverse thiaminases salvage different forms of degraded thiamin. We hypothesize that thiamine-I may initiate thiamin cleavage in a recently discovered thiamin catabolic pathway.

描述(申请人提供)：硫胺素是生命形式所必需的。硫胺素焦磷酸在碳水化合物的燃烧和支链氨基酸的合成中起着关键作用。硫胺素缺乏会导致疲劳、困惑、抑郁和易怒，如果不治疗，可能是致命的。硫胺素是由大多数细菌、真菌和植物生物合成的。人类必须从饮食中获取硫胺素。我们研究的总体目标是了解硫胺素生物合成和代谢的详细机制酶学。这些实验旨在填补我们对细菌和植物中的硫胺素嘧啶合成酶(Thic)、酵母中的硫胺素并噻唑合成酶(THI4)、酵母中的硫胺素嘧啶合成酶(THi5)以及硫胺素降解酶(硫胺酶)的了解的重要空白。THIC是一种新的自由基SAM酶，催化氨基咪唑核苷酸复杂重排为硫胺素嘧啶。我们最近确定了一种具有[4Fe-4S]团簇的ThIC结构。与典型的自由基SAM酶不同，簇结合结构域被拴在催化结构域上，并通过结构域交换插入到两个相关单体的活性部位。奇怪的是，在我们的初始结构中，该簇位于距离活性位点25�的位置，这表明结构域的移位必须在催化之前发生。酵母菌THI4催化NAD、甘氨酸和半胱氨酸缩合形成腺基化的羧基噻唑。我们已经证明THI4是一种铁依赖的自杀酶，而噻唑硫原子的来源是THI4本身的半胱氨酸侧链。THI4的结构确定了关键的活性部位残基，但我们还没有确定铁结合部位。在古生菌中，酵母菌THI4被证明催化1，5-二磷酸核糖异构化为1，5-二磷酸核酮糖。我们已经证明，古生菌的THI4同源基因也催化合成硫胺素噻唑，但使用硫化物作为硫源。我们还不知道这种双重活性是否存在于其他THI4‘S中。酵母THI5催化PLP和组氨酸的显著缩合形成硫胺素嘧啶。我们已经证明了tHi5是一种铁依赖的自杀酶，组氨酸侧链来自tHi5本身。THi5的结构确定了关键的活性部位残基，但我们尚未确定铁结合部位。对于Thic、THI4和tHi5，我们将确定底物、产物和类似物的结构，确定金属结合部位，并使用突变酶捕获中间体，从而填充关键的机制细节。硫胺素-I和硫胺素II是硫胺素降解酶，矛盾的是，它们经常与硫胺素生物合成酶聚集在一起。枯草杆菌硫胺酶-II参与了硫胺素的回收。因此，我们假设，回收降解的硫胺素可能是硫胺素酶-II的一般作用，不同的硫胺素酶回收不同形式的降解硫胺素。我们推测，硫胺素-I可能在最近发现的硫胺素分解代谢途径中启动硫胺素分解。