Functional diversification of the HD-superfamily; the Hydrolase/Oxygenase Dilemma

HD超家族的功能多样化；

基本信息

批准号：
9271972
负责人：
Maria-Eirini Pandelia
金额：
$ 24.58万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-05 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9271972
关键词：
Active Sites Address Adopted Affect Anabolism Antibiotics Antiviral Agents Binding Biochemical Bioinformatics Biological Assay Catabolism Chemicals Cleaved cell Competence Complications of Diabetes Mellitus Crystallization DNA Degradation Pathway Descriptor Electron Transport Complex III Employee Strikes Environment Enzymes Event Family Freezing Funding Glycine HIV HIV-1 Health Human Huntington gene Hydrolase Hydrolysis Immune response Inositol Insulin-Dependent Diabetes Mellitus Kinetics Lead Life Marines Mass Spectrum Analysis Measurement Measures Mediating Metalloproteins Metals Methods Modus Molecular Monitor Mono-S Mononuclear Mutagenesis Nature Nuclear Nucleotidases Nucleotides Oxidation-Reduction Oxygen Oxygenases Periodicity Phase Phosphorus Phylogenetic Analysis Physiological Play Property Proteins RNA Reaction Role Series Signal Transduction Source Structure Techniques Tertiary Protein Structure Therapeutic Agents Time Titrations Viral Work absorption analog base chemical reaction cofactor divalent metal experimental study inhibitor/antagonist inorganic phosphate inositol oxygenase insight member metalloenzyme microorganism novel overexpression phosphonate phosphoric diester hydrolase public health relevance screening small molecule structural genomics uptake

项目摘要

DESCRIPTION (provided by applicant): HD-domain proteins constitute a novel superfamily of metalloenzymes that counts presently more than 37,000 members in all three domains of life. Though there are generally annotated as as (phospho)hydrolases their functions are mostly unknown. Less than a decade ago, a novel diiron HD enzyme involved in the catabolism of inositol and associated with type I diabetes mellitus, namely myo-inositol oxygenase was demonstrated to carry out a radically different reaction using molecular oxygen to afford activation of its substrate. The only recently identified HD enzyme PhnZ, was also shown to follow the paradigm of MIOX, employing oxygen for the conversion of an organophosphonate to phosphate by marine microorganisms. The mechanism and structure of the reactive intermediates are presently unknown, but their mechanistic striking similarity to other nonheme Fe enzymes involved in the biosynthesis of antibiotics, invoke questions about the function of these enzymes and their possible implications on human health and environment. For this purpose, the first part of the project will focus on the characterization of PhnZ. This entails a combination of spectroscopic, structural, redox and activity studies so as to establish the modus operandii of such novel oxygenases. The substrate-free and bound forms of the enzyme will be studied so as to obtain for the first time combined structural and electronic information about the 'on' and 'off' reactive states for which crystallographic information has been extremely challenging, how substrate or inhibitors tune and affect the properties of the active site of the enzyme. This information will set the grounds for the discovery of compounds that can activate and inhibit these enzymes, therefore providing powerful control over their function. In addition to, the characterization of downstream events in the chemical reactions will likely establish the common strategy that specific nonheme Fe enzymes (mononuclear or dinuclear) adopt to carry out difficult and environmentally important reactions. The second part of the project aims at mapping the catalytic landscape of (dinuclear) HD domain enzymes, discovery of new functions and identifying the type and the role of metals in modulating specific activities (hydrolysis vs oxygenation). For this purpose, on the basis of phylogenetic analysis new attractive protein targets of unknown function have been identified. Selected protein will be overexpressed and purified. These will be spectroscopically characterized with a combination of EPR, M�ssbauer, crystallographic and NMR techniques. A profile of their activities will be established by screening activities for specific substrates and mass spectrometry methods. Presently there are a handful of HD domain enzymes implicated in immunoresponse, such as restriction factors for HIV-1 or nucleotidases attacking viral nucleotides that have come into the scientific focus. Their function is not completely understood, whereas the presence of one or two metals is not known whether it is functional, structural or co-catalytic. This work will begin during the K99 funding period and will continue during the independent phase and will attempt to study these enzymes and draw the molecular background of their function (hydrolytic vs oxygenation). The long-range purpose of this second phase of the project is to establish on the basis of bioinformatics, crystallographic, mutagenesis and activity studies the determinants directing distinct functions within the HD superfamily that will ultimately lead to the identification new antiviral factors and therapeutic agents as well as the discovery of novel oxygenases implicated in chemically difficult small molecule transformations.

描述（由申请人提供）：HD 结构域蛋白构成了一个新的金属酶超家族，目前在生命的所有三个领域都有超过 37,000 个成员，尽管它们通常被注释为（磷酸）水解酶，但它们的功能大多未知。十年前，一种新型二铁 HD 酶参与肌醇的分解代谢并与 I 型糖尿病相关，即肌醇加氧酶最近发现的唯一的 HD 酶 PhnZ 被证明可以进行完全不同的反应，以激活其底物，也被证明遵循 MIOX 的范例，利用氧气通过海洋微生物将有机膦酸盐转化为磷酸盐。反应中间体的机制和结构目前尚不清楚，但它们的机制与抗生素生物合成中涉及的其他非血红素铁酶具有惊人的相似性，引发了有关这些酶的功能及其可能对抗生素的影响的问题。为此，该项目的第一部分将重点关注 PhnZ 的表征，这需要结合光谱、结构、氧化还原和活性研究，以确定此类新型加氧酶的底物。 -将研究酶的游离形式和结合形式，以便首次获得有关该酶的组合结构和电子信息晶体学信息非常具有挑战性的“开”和“关”反应状态，底物或抑制剂如何调整和影响酶活性位点的特性，这些信息将为发现可以激活和关闭的化合物奠定基础。抑制这些酶，从而对其功能提供强有力的控制此外，化学反应中下游事件的表征可能会建立特定非血红素铁酶（单核或双核）用于进行困难且对环境重要的反应的共同策略。 .第二个该项目的一部分旨在基于系统发育，绘制（双核）HD 域酶的催化图谱，发现新功能并确定金属在调节特定活性（水解与氧化）中的类型和作用。已鉴定出具有未知功能的新的有吸引力的蛋白质靶标，这些蛋白质将通过 EPR、穆斯堡尔、晶体学和 NMR 技术的组合进行光谱表征。将通过筛选特定底物的活性和质谱方法来确定它们的活性概况。目前，有一些与免疫反应有关的 HD 结构域酶，例如 HIV-1 的限制因子或攻击病毒核苷酸的核苷酸酶。它们的功能尚不完全清楚，而其中一种或两种金属的存在是否具有功能性、结构性或协同催化作用尚不清楚。这项工作将在 K99 资助期间开始并将继续进行。在独立阶段，我们将尝试研究这些酶并绘制它们功能的分子背景（水解与氧化）。该项目第二阶段的长期目标是建立在生物信息学、晶体学、诱变和作用的基础上。活动研究指导 HD 超家族内不同功能的决定因素，最终将导致识别新的抗病毒因子和治疗剂以及发现涉及化学上困难的小分子转化的新型加氧酶。