Nonheme diiron enzymes: understanding oxygen activation in human deoxyhypusine hy

描述(申请人提供)：非血红素二铁酶参与许多生物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，以及从RNA创建DNA构建块。这些酶的催化循环涉及O2的结合和随后的激活，以进行广泛的化学作用。重要的是，这些酶的作用机制被认为依赖于一种常见的(？-1，2-过氧基)二铁中间体的形成。这种结构被认为是稳定的，需要活化到更具活性的中间体，以便随后的催化。虽然这个酶超家族已经被研究了很多年，但关于酶如何激活常见的过氧化中间体以保持如此多样化的反应活性的几个关键机制细节尚不清楚。在不同的蛋白质折叠和双铁配位环境中发现新的双铁酶促使人们进一步研究蛋白质和双铁位点在过氧化反应形成和控制中的作用。特别是，QUE实验室在人脱氧亚硫氨酸羟基酶(HDOHH)中发现了过氧中间体。HDOHH催化氨基酸次生氨酸的翻译后羟化。这种氨基酸只存在于真核细胞翻译起始因子5A(EIF5A)中，是细胞增殖所必需的，使hDOHH成为癌症和艾滋病毒治疗的诱人靶点。HDOHHperoxo中间体(HDOHperoxo)可稳定数天，使其成为迄今发现的寿命最长的过氧物物种。此外，二铁簇位于蛋白质折叠和协调环境中，这一环境有别于所有其他非血红素二铁酶。该项目建议使用一系列生化和光谱技术来研究调节hDOHperoxo激活的机制和结构细节。具体地说，这项建议将调查底物结合、pH和独特的二铁配位环境如何有助于hDOHperoxo的形成和激活。HDOHperoxo的生成速率随底物浓度、不同pH值和氢化反应的变化将用紫外可见光谱进行测量。四种谷氨酸活性中心配体(Glu57、Glu90、Glu208、Glu241)对天冬氨酸和谷氨酰胺的单独诱变作用将被用来评估静电和立体物质对hDOHperoxo稳定性的贡献。活性中心结构的电子和几何细节 作为上述修饰(底物、pH、配位配体)的函数，将使用共振拉曼光谱、穆斯堡尔光谱和XAS光谱来确定。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基物种来进行。从这些实验中获得的信息将增加我们对影响过氧化中间体反应活性的特征的知识，特别是在新的蛋白质折叠和不同的双铁配位环境中发现的特征，进一步加深我们对O-O键激活是如何完成的理解，并可能导致更好地理解如何将hDOHH靶向癌症和艾滋病毒治疗。

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.