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

描述（由申请人提供）：本项目检查了FeII/？-酮戊二酸（？KG）依赖的羟化酶，并探讨它们催化的反应的多样性。该酶家族的成员广泛存在于细菌和真核生物（包括人类）中，它们促进具有根本重要性的反应，包括DNA/RNA修复、大量小分子的合成/降解、脂质代谢以及与氧传感、染色质脱甲基或结构相互作用相关的蛋白质羟基化。本建议中详细介绍的研究侧重于四个目标。首先，我们将通过将创新的连续流拉曼光谱方法应用于TauD（该酶家族中研究得最好的成员）来定义早期催化过程中的化学步骤。特别感兴趣的将是一个关键的TauD变体，慢慢形成已知的FeIV=O中间体的属性，以及嗜热同系物的行为。平行研究将探索在其他两个可用的家族成员中鉴别的反应中间体的均匀性。第二，脉冲EPR技术将被用来调查的几何形状的活性位点环境的酶结合一氧化氮（NO），O2的替代品。使用这些新方法的测量将与TauD，我们有晶体学信息进行验证，然后应用于XanA，一种黄嘌呤降解酶，缺乏结构数据。特别是，这些技术将被用来探测小的结构变化，在活性位点底物结合后或在选定的变体蛋白质。第三，将确认TauD中第二个FeII结合位点的存在，并研究该位点的功能。作为这些研究的一部分，我们将探索使用磷光猝灭，以获得厌氧蛋白质的热力学结合数据。最后，生化和光谱特性将被阐明TET 1，5-meC羟化酶，可能与另一种酶作为DNA去甲基化酶的功能。总的来说，这项工作将提高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作用。这些研究具有医学相关性，因为了解这种机制对于开发与FeII/？KG羟化酶，用于防御病原体，其中这些酶发挥重要作用，并用于优化这些酶在其他微生物中合成抗生素。

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.