Mechanism of taurine: alpha-ketoglutarate dioxygenase

牛磺酸的作用机制：α-酮戊二酸双加氧酶

• 批准号: 7334215
• 负责人: CARSTEN KREBS
• 金额: $ 25.11万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7334215
• 关键词: Achievement; Acids; Bacteria; Binding; Carbon; Catalysis; Chemicals; Class; Cleaved cell; Collagen; Complement component C1s; Complex; Condition; DNA Alkylation; Data; Decarboxylation; Dioxygen; Dioxygenases; Disease; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electronics; Electrons; Enzymes; Escherichia; Ethane; Event; Fatty Acids; Freezing; Functional disorder; Goals; Human; Hydroxylation; Hypoxia; Investigation; Iron; Isotopes; Kinetics; Knowledge; Label; Laboratories; Ligands; Mammals; Measures; Methods; Molecular; Monitor; Monobactams; Object Attachment; Organism; Oxygen; Pathway interactions; Pentanes; Positioning Attribute; Process; Property; Range; Rate; Reaction; Relative (related person); Research Project Grants; Roentgen Rays; Role; Sampling; Site; Spectrum Analysis; Structure; Sulfonic Acids; System; Taurine; Techniques; Temperature; Time; Variant; Work; absorption; alpha ketoglutarate; analog; complex IV; insight; irradiation; novel; oxidation; pentane; repaired; research study; response; taurine-alpha-ketoglutarate dioxygenase

DESCRIPTION (provided by applicant) The Fe(ll)-/a-ketoglutarate-dependent dioxygenases couple oxidative decarboxylation of a-ketoglutarate with hydroxylation of unactivated carbon atoms on a variety of substrates. Enzymes in this class catalyze important reactions in organisms from bacteria to humans and have essential roles in such critical processes as sensing of oxygen and response to hypoxia in mammals; synthesis of B-lactam antibiotics, collagen, and fatty acids in a range of organisms; and repair of DNA alkylation damage in bacteria and humans. In addition, dysfunction of enzymes in the class of enzymes has been associated with numerous disease states. It has been proposed that these enzymes operate by a common mechanism. Despite extensive investigation in many laboratories, there had been, until our recent work, no direct evidence for any of the several intermediate states proposed to follow after reaction of the enzyme with molecular oxygen. We have recently obtained the first direct evidence for an oxidized iron intermediate in the reaction catalyzed by one enzyme in this class, taurine/a-ketoglutarate dioxygenase (TauD), and have shown that the novel complex accumulates to levels sufficient for a thorough spectroscopic characterization. Preliminary data indicate that the intermediate has a formal Fe(IV) oxidation state and suggest two most likely structures for the complex. The main objectives of this research project are: (1) To place the novel Fe(IV) intermediate in the sequence of events leading to O2 activation, a-ketoglutarate oxidative decarboxylation, and taurine hydroxylation by a combination of rapid kinetics experiments in order to limit possible structural assignments and choose between the two most likely structures; (2) To rigorously characterize this novel reaction intermediate by a combination of spectroscopic techniques in order to elucidate its electronic and geometric structure; and (3) To use modified reactants (substrate analogues and site directed TauD variants) to accumulate and then trap and characterize intermediate states in the reaction pathway that are not accessible in the reaction with the normal components. Achievement of these goals will provide new and unprecedented insight into the molecular details of the catalytic mechanism of TauD and, more importantly, of the Fe(ll)/a-ketoglutarate-dependent dioxygenases in general.

Fe(ll)-/a-酮戊二酸依赖的双加氧酶将a-酮戊二酸的氧化脱羧与各种底物上的非活性碳原子的羟基化结合起来。这类酶催化从细菌到人类的重要反应，在哺乳动物的氧气感知和缺氧反应等关键过程中发挥重要作用；在一系列生物体中合成b -内酰胺类抗生素、胶原蛋白和脂肪酸；以及修复细菌和人类的DNA烷基化损伤。此外，这类酶的功能障碍与许多疾病状态有关。有人提出这些酶是通过一种共同的机制起作用的。尽管在许多实验室进行了广泛的研究，但直到我们最近的工作，还没有直接证据表明酶与分子氧反应后会出现几种中间状态。我们最近获得了第一个直接证据，证明在这类酶中牛磺酸/a-酮戊二酸双加氧酶（TauD）催化的反应中存在氧化铁中间体，并表明这种新型复合物积累到足以进行彻底的光谱表征的水平。初步数据表明中间体具有正式的Fe（IV）氧化态，并提出了两种最可能的配合物结构。本研究项目的主要目标是：(1)通过结合快速动力学实验，将新型Fe（IV）中间体置于导致O2活化、a-酮戊二酸氧化脱羧和牛磺酸羟基化的事件序列中，以限制可能的结构分配并在两种最可能的结构之间进行选择；(2)结合光谱技术对这种新型反应中间体进行严格表征，以阐明其电子和几何结构；(3)使用修饰的反应物（底物类似物和定点TauD变体）积累，然后捕获和表征反应途径中的中间状态，这些中间状态在与正常组分的反应中是无法获得的。这些目标的实现将为TauD催化机制的分子细节提供新的和前所未有的见解，更重要的是，一般来说，Fe(ll)/a-酮戊二酸依赖的双加氧酶。