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

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/β-酮戊二酸 (?KG) 依赖性羟化酶所使用的酶机制，并探索了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，它们促进至关重要的反应，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相关的蛋白质羟基化，染色质去甲基化或结构相互作用。该提案中详细研究的重点是四个目标。首先，我们将通过对 TauD（该酶家族中研究最充分的成员）应用创新的连续流拉曼光谱方法来定义早期催化过程中的化学步骤。特别令人感兴趣的是缓慢形成已知 FeIV=O 中间体的关键 TauD 变体的特性，以及嗜热同系物的行为。平行研究将探讨其他两个可用家族成员中已识别的反应中间体的一致性。其次，脉冲 EPR 技术将用于研究与一氧化氮（NO）（O2 的替代物）结合的酶的活性位点环境的几何形状。使用这些新方法进行的测量将通过 TauD 进行验证，我们拥有晶体学信息，然后应用于 XanA（一种黄嘌呤降解酶），但缺乏结构数据。特别是，这些技术将用于探测底物结合时活性位点或选定的变体蛋白质中的微小结构变化。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来获得厌氧蛋白质的热力学结合数据。最后，TET1 的生化和光谱特性将得到阐明，TET1 是一种 5-meC 羟化酶，可能与另一种酶一起发挥 DNA 脱甲基酶的作用。总的来说，这项工作将增强我们对这种多功能酶家族常见酶机制的理解，同时进一步为其各个成员定义新的、多样化的角色。此类研究具有医学意义，因为了解这一机制对于开发与 FeII/?KG 羟化酶缺陷相关的人类遗传疾病的治疗方法、防御此类酶发挥重要作用的病原体以及优化这些酶的抗生素合成至关重要。在其他微生物中。