Structural Studies of Enzymes of Thiamin Biosynthesis

硫胺素生物合成酶的结构研究

• 批准号: 8759058
• 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/8759058
• 关键词: 5' -deoxyadenosine; Active Sites; Address; Amino Acids; Anabolism; Antibiotics; Archaea; Area; Bacillus (bacterium); Bacillus subtilis; Bacteria; Beriberi; Binding; Binding Sites; Biochemical; Branched-Chain Amino Acids; Carbohydrates; Catabolism; Catalysis; Catalytic Domain; Collaborations; Complex; Confusion; Consumption; Cysteine; DNA Sequence Rearrangement; 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 Å 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（由申请人提供）：硫胺素是生命形式所必需的。焦磷酸硫胺素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、混乱、抑郁和易怒，如果不及时治疗，可能是致命的。硫胺素由大多数细菌、酵母、真菌和植物生物合成。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酶（ThiC），酵母中的硫胺素噻唑合酶（THI 4），酵母中的硫胺素嘧啶合酶（THI 5）和硫胺素降解酶（硫胺素酶）的理解中的重要空白。ThiC是一种新型的自由基SAM酶，催化氨基咪唑核苷酸重排为硫胺嘧啶。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，簇结合结构域与催化结构域相连，并通过结构域交换插入到双重相关单体的活性位点中。奇怪的是，在我们的初始结构中，该簇位于距离活性位点25 μ m处，这表明结构域的移位必须在催化之前发生。酵母THI 4催化NAD、甘氨酸和半胱氨酸缩合形成腺苷酸化羧基噻唑。我们已经证明THI 4是一种铁依赖性自杀酶，噻唑硫原子的来源是THI 4本身的半胱氨酸侧链。THI 4的结构确定了关键活性位点残基，但我们尚未确定铁结合位点。在古细菌中，酵母THI 4直向同源物显示催化1，5-二磷酸核糖异构化为1，5-二磷酸核酮糖。我们已经表明，古细菌THI 4直系同源物也催化硫胺噻唑的合成，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THI 4中。酵母THI 5催化PLP和组氨酸的显著缩合以形成硫胺嘧啶。我们已经证明THI 5是一种铁依赖性自杀酶，组氨酸侧链来自THI 5本身。THI 5的结构确定了关键活性位点残基，但我们尚未确定铁结合位点。对于ThiC，THI 4和THI 5，我们将确定底物，产物和类似物的结构，确定金属结合位点，并使用突变酶捕获中间体，从而填充关键的机械细节。硫胺素酶-I和II是硫胺素降解酶，其矛盾地经常与硫胺素生物合成酶聚集。枯草芽孢杆菌硫胺素酶-II被证明参与硫胺素补救。因此，我们推测，补救降解硫胺素可能是硫胺素酶-II的一般作用，不同的硫胺素酶补救不同形式的降解硫胺素。我们推测，硫胺素-I可能会启动硫胺素裂解在最近发现的硫胺素分解代谢途径。