Mechanism of taurine: alpha-ketoglutarate dioxygenase

说明书(由申请人提供)铁(11)-/a-酮戊二酸依赖的双加氧酶将a-酮戊二酸的氧化脱羧基与各种底物上未活化的碳原子的羟基化结合在一起。这类酶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对低氧的反应；一系列生物体中B-内酰胺抗生素、胶原和脂肪酸的合成；以及细菌和人类DNA烷基化损伤的修复等关键过程中发挥重要作用。此外，酶类中的酶功能障碍与许多疾病状态有关。有人提出，这些酶的作用机制是共同的。尽管在许多实验室进行了广泛的研究，但在我们最近的工作之前，没有直接证据表明酶与分子氧反应后可能出现的几种中间状态中的任何一种。我们最近首次获得了在这类酶-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的直接证据，并表明这种新的络合物积累到足以进行彻底光谱表征的水平。初步数据表明，中间体具有正式的Fe(IV)氧化态，并暗示了该络合物的两种最可能的结构。本研究项目的主要目标是：(1)通过快速动力学实验的组合，将新的Fe(IV)中间体置于导致O2活化、α-酮戊二酸氧化脱羧基和牛磺酸羟基化的一系列事件中，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，以阐明其电子结构和几何结构；以及(3)使用修饰的反应物(底物类似物和定点定向的TauD变体)来积累，然后捕获和表征反应路径中无法与正常组分反应的中间状态。这些目标的实现将为了解TauD的催化机制提供前所未有的新的分子细节，更重要的是，总体上将提供依赖Fe(11)/a-酮戊二酸的双加氧酶的分子细节。

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.