Characterization of Fe(II)/alpha-ketoglutarate-dependent hydroxylases

描述(由申请人提供)：本项目研究依赖FeII/？-酮戊二酸(？kg)的羟基酶使用的酶机制，并探索它们催化的反应的多样性。这个酶家族的成员广泛存在于细菌和真核生物(包括人类)中，它们促进基本的反应，包括DNA/RNA修复，大量小分子的合成/降解，脂代谢，以及与氧感知、染色质去甲基化或结构相互作用有关的蛋白质羟化。这项建议中详细说明的研究集中在四个目标上。首先，我们将通过应用创新的连续流动拉曼光谱方法来定义早期催化过程中的化学步骤，TauD是该酶家族中研究最好的成员。特别令人感兴趣的是一个关键的TauD变体的性质，它慢慢形成已知的FeIV=O中间体，以及嗜热同系物的行为。平行研究将探索在另外两个可用的家族成员中确定的反应中间体的一致性。其次，脉冲EPR技术将被用来研究结合一氧化氮(NO)的酶的活性部位环境的几何形状，NO是O2的替代品。使用这些新方法的测量将用TauD进行验证，在那里我们有结晶学信息，然后应用于Xana，一种缺乏结构数据的黄嘌呤降解酶。特别是，这些技术将被用来探测底物结合时活性部位的微小结构变化或在选定的变异蛋白中。第三，将确认在TauD中存在第二个FeII结合位点，并将研究该结合位点的功能。作为这些研究的一部分，我们将探索使用磷光猝灭来获得厌氧蛋白质的热力学结合数据。最后，将阐明TET1的生化和光谱性质，它是一种5-MEC羟基酶，可能与另一种酶一起作为DNA去甲基酶发挥作用。总之，这项工作将加深我们对这个多功能酶家族共同的酶机制的理解，同时进一步定义其单个成员的新的和不同的角色。这类研究具有医学意义，因为了解这一机制对于开发与FeII/kg羟基酶缺陷相关的人类遗传病的治疗方法至关重要，对于防御这些酶起关键作用的病原体，以及优化这些酶在其他微生物中合成抗生素至关重要。

DESCRIPTION (provided by applicant): This project examines the enzymatic mechanism used by FeII/?-ketoglutarate (?KG)-dependent hydroxylases and explores the diversity of reactions they catalyze. Members of this enzyme family are widespread in bacteria and eukaryotes (including humans) where they promote reactions of fundamental importance including DNA/RNA repair, synthesis/degradation of a vast repertoire of small molecules, lipid metabolism, and protein hydroxylation related to oxygen sensing, chromatin demethylation, or structural interactions. The studies detailed in this proposal focus on four aims. First, we will define the chemical steps during early catalysis by applying an innovative continuous- flow Raman spectroscopic approach to TauD, the best studied member of this enzyme family. Of special interest will be the properties of a key TauD variant that slowly forms the known FeIV=O intermediate, as well as the behavior of a thermophilic homologue. Parallel studies will probe for uniformity of the identified reaction intermediates in two other available family members. Second, pulsed EPR techniques will be utilized to investigate the geometries of active site environments for enzymes with bound nitric oxide (NO), a surrogate of O2. Measurements using these novel methods will be validated with TauD, where we have crystallographic information, and then applied to XanA, a xanthine-degrading enzyme, for which structural data are lacking. In particular, these techniques will be exploited to probe small structural changes at the active site upon substrate binding or in selected variant proteins. Third, the presence of a second FeII binding site in TauD will be confirmed and the function of this site will be investigated. As part of these studies, we will explore the use of phosphorescence quenching to obtain thermodynamic binding data on anaerobic proteins. Finally, biochemical and spectroscopic properties will be elucidated for TET1, a 5-meC hydroxylase that might function with another enzyme as a DNA demethylase. In total, this work will enhance our understanding of the enzyme mechanism common to this versatile enzyme family while further defining new and diverse roles for its individual members. Such studies have medical relevance because understanding of this mechanism is critical for developing treatments of human genetic diseases associated with defects in FeII/?KG hydroxylases, for defending against pathogens where such enzymes play essential roles, and for optimizing the synthesis of antibiotics by these enzymes in other microbes.