Nonheme diiron enzymes: understanding oxygen activation in human deoxyhypusine hy

非血红素二铁酶：了解人脱氧马匹素 hy 中的氧活化

• 批准号: 8765622
• 负责人: Lisa Engstrom
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2015-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765622
• 关键词: Active Sites; Address; Affect; Amino Acids; Anabolism; Antibiotics; Binding; Biochemical; Biological; Catalysis; Cell Proliferation; Chemicals; Chemistry; DNA; Dioxygen; Electronics; Electrostatics; Environment; Enzymes; Equipment and supply inventories; Eukaryotic Cell; Glutamine; Goals; HIV; Health; Housing; Human; Hydroxylation; Individual; Investigation; Laboratories; Lead; Life; Ligands; Light; Malignant Neoplasms; Measures; Methanol; Modification; Mutagenesis; Mutate; Nature; Nitrogen; Outcome; Oxygen; Peptide Initiation Factors; Process; Proteins; Protons; RNA; Reaction; Research; Research Proposals; Role; Site; Solvents; Spectrum Analysis; Structure; System; Techniques; Therapeutic; Translating; carboxylate; deoxyhypusine; deoxyhypusine monooxygenase; enzyme mechanism; hypusine; insight; novel; protein folding; protonation; research study; small molecule; time use

DESCRIPTION (provided by applicant): Nonheme diiron enzymes are involved in the synthesis of many biologically and industrially relevant compounds, including the formation of methanol, the biosynthesis of antibiotics, and the creation of DNA building blocks from RNA. The catalytic cycles of these enzymes involve the binding and subsequent activation of O2 to carry out a wide variety of chemistry. Importantly, the mechanism of these enzymes is thought to depend on the formation of a common (¿-1,2-peroxo)diferric intermediate. This structure is proposed to be stable, requiring activation to a more reactive intermediate for subsequent catalysis. Although this enzyme superfamily has been studied for many years, several key mechanistic details surrounding how enzymes activate the common peroxo intermediate to maintain such diverse reactivity are unknown. The identification of new diiron enzymes within distinct protein folds and diiron coordination environments has prompted further investigation into the role of both the protein and the diiron site on formation and control of peroxo reactivity In particular, the Que laboratory has identified a peroxo intermediate in human deoxyhypusine hydroxylase (hDOHH). hDOHH catalyzes the post-translational hydroxylation of the amino acid hypusine. This amino acid is found only in the eukaryotic translational initiation factor 5A (eIF5A and is required for cell proliferation, making hDOHH an appealing target for cancer and HIV treatments. The hDOHH peroxo intermediate (hDOHHperoxo) is stable for days, making it the longest-lived peroxo species identified to date. Furthemore, the diiron cluster is housed in a protein fold and coordination environment unique from all other nonheme diiron enzymes. This project proposes to investigate mechanistic and structural details that regulate hDOHHperoxo activation using a host of biochemical and spectroscopic techniques. Specifically, this proposal will investigate how substrate binding, pH, and the distinct diiron coordination environment contribute to the formation and activation of hDOHHperoxo. The rates of hDOHHperoxo formation as a function of substrate concentration, varied pH and deuteration will be measured using UV-visible spectroscopy. Individual mutagenesis of four active site Glu ligands (Glu57, Glu90, Glu208, Glu241) to Asp and Gln will be performed to assess the contribution of electrostatics and sterics on hDOHHperoxo stability. Electronic and geometric details of the active site structure as a function of the above modifications (substrate, pH, coordinating ligand) will be ascertained using resonance Raman, M¿ssbauer, and XAS spectroscopies. Importantly, these studies will be carried out for the first time using a peroxo species competent in carrying out the native chemistry of the enzyme. Information obtained from these experiments will add to our knowledge of features that affect the reactivity of peroxo intermediates, particularly those found in new protein folds and distinct diiron coordination environments, furthering our understanding of how O-O bond activation is accomplished and may lead to a better understanding of how to target hDOHH for cancer and HIV therapeutics.

描述（由申请人提供）：非血红素二铁酶参与许多生物学和工业相关化合物的合成，包括甲醇的形成、抗生素的生物合成和由RNA产生DNA结构单元。这些酶的催化循环涉及O2的结合和随后的活化，以进行各种各样的化学反应。重要的是，这些酶的机制被认为取决于一个共同的（戊-1，2-过氧）二铁中间体的形成。该结构被认为是稳定的，需要活化为更反应性的中间体用于随后的催化。虽然这个酶超家族已经被研究了很多年，但围绕酶如何激活常见的过氧中间体以保持如此多样的反应性的几个关键机制细节是未知的。在不同的蛋白质折叠和二铁配位环境中鉴定新的二铁酶，促使进一步研究蛋白质和二铁位点对过氧反应性的形成和控制的作用。特别是，Que实验室已经鉴定了人脱氧羟腐胺赖氨酸羟化酶（hDOHH）中的过氧中间体。hDOHH催化氨基酸羟腐胺赖氨酸的翻译后羟基化。该氨基酸仅在真核翻译起始因子5A（eIF 5A）中发现，并且是细胞增殖所需的，使得hDOHH成为癌症和HIV治疗的有吸引力的靶标。hDOHH过氧中间体（hDOHH peroxo）可稳定数天，使其成为迄今为止发现的寿命最长的过氧物质。此外，二铁簇被安置在一个蛋白质折叠和协调环境独特的所有其他nonheme二铁酶。该项目提出了调查机制和结构的细节，调节hDOHH过氧活化使用主机的生化和光谱技术。具体而言，该提案将调查如何基板结合，pH值，和独特的二铁配位环境有助于形成和激活的hDOHH过氧。将使用UV-可见光谱法测量作为底物浓度、不同pH和氘化的函数的hDOHH过氧形成速率。将进行四个活性位点Glu配体（Glu 57、Glu 90、Glu 208、Glu 241）对Asp和Gln的单独诱变，以评估静电和空间效应对hDOHH过氧稳定性的贡献。活性中心结构的电子和几何细节 将使用共振拉曼、穆斯堡尔和XAS光谱确定作为上述修饰（底物、pH、配位配体）的函数。重要的是，这些研究将首次使用能够进行酶的天然化学的过氧物种进行。从这些实验中获得的信息将增加我们对影响过氧中间体反应性的特征的了解，特别是在新的蛋白质折叠和不同的二铁配位环境中发现的那些，进一步了解O-O键活化是如何完成的，并可能导致更好地了解如何靶向hDOHH用于癌症和HIV治疗。