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

Fe(II)/α-酮戊二酸依赖性羟化酶的表征

• 批准号: 8538998
• 负责人: ROBERT P HAUSINGER
• 金额: $ 36万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538998
• 关键词: Active Sites; Antibiotics; Bacteria; Behavior; Binding; Binding Sites; Biochemical; Biological; Catalysis; Chemicals; Chromatin; Collaborations; Comparative Study; DNA; DNA Methyltransferase; DNA Modification Methylases; Data; Defect; Development; Dioxygenases; Drug Controls; Drug Targeting; Electrons; Engineering; Environment; Enzymes; Eukaryota; Family; Family member; Goals; Hereditary Disease; Homologous Gene; Human; Human Genetics; Hydroxylation; Individual; Infection; Kinetics; Laboratories; Lead; Ligand Binding; Ligands; Measurement; Medical; Metals; Methods; Microbe; Mixed Function Oxygenases; Molecular; Nitric Oxide; Outcome Study; Oxygen; Pathway interactions; Physiologic pulse; Play; Property; Proteins; RNA; Raman Spectrum Analysis; Reaction; Resolution; Role; Sampling; Site; Spectrum Analysis; Structure; Taurine; Techniques; Testing; Thermodynamics; Thymine; Variant; Work; Xanthines; alpha ketoglutarate; antimicrobial drug; catalyst; cold temperature; cryogenics; demethylation; enzyme mechanism; experience; improved; innovation; interest; lipid metabolism; member; microorganism; mutant; novel; pathogen; phosphorescence; repaired; small molecule; thermophilic bacteria; tool; two-dimensional; Active Sites; Antibiotics; Bacteria; Behavior; Binding; Binding Sites; Biochemical; Biological; Catalysis; Chemicals; Chromatin; Collaborations; Comparative Study; DNA; DNA Methyltransferase; DNA Modification Methylases; Data; Defect; Development; Dioxygenases; Drug Controls; Drug Targeting; Electrons; Engineering; Environment; Enzymes; Eukaryota; Family; Family member; Goals; Hereditary Disease; Homologous Gene; Human; Human Genetics; Hydroxylation; Individual; Infection; Kinetics; Laboratories; Lead; Ligand Binding; Ligands; Measurement; Medical; Metals; Methods; Microbe; Mixed Function Oxygenases; Molecular; Nitric Oxide; Outcome Study; Oxygen; Pathway interactions; Physiologic pulse; Play; Property; Proteins; RNA; Raman Spectrum Analysis; Reaction; Resolution; Role; Sampling; Site; Spectrum Analysis; Structure; Taurine; Techniques; Testing; Thermodynamics; Thymine; Variant; Work; Xanthines; alpha ketoglutarate; antimicrobial drug; catalyst; cold temperature; cryogenics; demethylation; enzyme mechanism; experience; improved; innovation; interest; lipid metabolism; member; microorganism; mutant; novel; pathogen; phosphorescence; repaired; small molecule; thermophilic bacteria; tool; two-dimensional

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.

描述（由申请人提供）：该项目研究了FeII/？ - 酮谷甲酸酯（？kg）依赖性羟基酶的酶促机制，并探讨了它们催化的反应的多样性。该酶家族的成员在细菌和真核生物（包括人类）中广泛促进了基本重要性的反应，包括DNA/RNA修复，合成/降解小分子的大量曲目，脂质代谢代谢和蛋白质羟基化与氧化基因的相互作用，磷酸化的相互作用。该提案中详细介绍的研究重点是四个目标。首先，我们将通过将创新的连续流拉曼光谱方法应用于该酶家族的最佳成员Taud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变体的特性，该变体慢慢形成已知的Feiv = O中间体以及嗜热同源物的行为。平行研究将探究其他两个可用家庭成员中确定的反应中间体的均匀性。其次，将利用脉冲EPR技术来研究具有结合一氧化氮（NO）的酶的活性位点环境的几何形状，替代O2。使用这些新方法的测量将用Taud进行验证，其中我们拥有晶体学信息，然后应用于Xana，一种降解酶，为此缺乏结构性数据。特别是，这些技术将被利用以探测底物结合或选定变体蛋白质中活性位点的微小结构变化。第三，将确认TAUD中第二个FEII结合位点的存在，并将研究该站点的功能。作为这些研究的一部分，我们将探索使用磷光淬灭以获得厌氧蛋白上的热力学结合数据。最后，将为TET1阐明生化特性和光谱特性，TET1是一种5-MEC羟化酶，可能与另一种酶一起用作DNA脱甲基酶。总的来说，这项工作将增强我们对这个多功能酶家族常见的酶机制的理解，同时进一步为其成员定义了新的和多样化的作用。此类研究具有医学相关性，因为对这种机制的理解对于开发与FeII/？kg羟基酶缺陷相关的人类遗传疾病的治疗至关重要，对于这种酶在其他微生物中通过这些酶来优化抗生素合成的病原体，以防御病原体。