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: 6837204
• 负责人: CARSTEN KREBS
• 金额: $ 26.49万
• 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 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6837204
• 关键词: Mossbauer spectrometry; X ray spectrometry; alpha ketoglutarate; carboxylation; catalyst; chemical reaction; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; oxygenases; taurine

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.

描述（由申请人提供） Fe(II)-/α-酮戊二酸依赖性双加氧酶将α-酮戊二酸的氧化脱羧与多种底物上未活化碳原子的羟基化偶联。此类酶可催化从细菌到人类的生物体中的重要反应，并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，酶类中的酶功能障碍与多种疾病状态相关。有人提出这些酶通过共同的机制起作用。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为止，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，证明在此类酶（牛磺酸/α-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光谱表征的水平。初步数据表明该中间体具有形式的 Fe(IV) 氧化态，并表明该配合物有两种最可能的结构。该研究项目的主要目标是：（1）通过快速动力学实验的结合，将新型 Fe(IV) 中间体置于导致 O2 活化、α-酮戊二酸氧化脱羧和牛磺酸羟基化的事件序列中，以便限制可能的结构分配并在两个最可能的结构之间进行选择； （2）结合光谱技术严格表征这种新型反应中间体，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）来积累，然后捕获和表征反应途径中的中间态，这些中间态在与正常组分的反应中无法达到。这些目标的实现将为 TauD 催化机制的分子细节提供新的、前所未有的见解，更重要的是，Fe(II)/α-酮戊二酸依赖性双加氧酶的分子细节。