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

HD超家族的功能多样化；

• 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
• 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

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（由适用提供）：HD域蛋白构成了金属酶的新型超家族，在生命的所有三个领域中都表现出37,000多名成员。尽管通常有注释为AS（磷）水解酶它们的功能大多未知。不到十年前，一种新型的Diron HD酶参与肌醇的分解代谢和与I型糖尿病相关的一种，即肌醇肌酸氧酶，即使用分子氧进行根本的不同反应，以实现其底物的激活。最近唯一发现的HD酶PHNZ也显示出遵循Miox的范式，该氧气利用氧气通过海洋微生物将有机膦酸酯转化为磷酸盐。反应性中间体的机制和结构尚不清楚，但是它们与涉及抗生素生物合成的其他非血红素Fe酶的机械惊人相似性，引用了有关这些酶功能及其对人类健康和环境的可能影响的问题。为此，项目的第一部分将重点放在PHNZ的特征上。该组织是光谱，结构，氧化还原和活性研究的结合，以建立这种新型氧合酶的作案。酶的无基质和结合形式将进行研究，以便首次获得有关该酶的结构和电子信息 晶体学信息受到极为挑战的“在”和“ OFF”反应状态，底物或抑制剂如何调整并影响酶的活性位点的性质。这些信息将为发现可以激活和抑制这些酶的化合物树立理由，从而为其功能提供强大的控制。此外，化学反应中下游事件的表征可能会确定特定的非血红素Fe酶（单核或核酸酶）采用的常见策略，以进行困难和环境重要的反应。该项目的第二部分旨在绘制（Dinuclear）HD域酶的催化景观，发现新功能并确定金属在调节特定活动中的类型和作用（水解与氧化）。为此，在系统发育分析的基础上，已经确定了未知功能的新有吸引力的蛋白质靶标。选定的蛋白质将过表达和纯化。这些将通过EPR，MSSBAUER，晶体学和NMR技术的组合来表征光谱。通过筛选特定底物和质谱法的筛选活动，将确定其活动的概况。目前，在免疫响应中实施了少数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，这些因素已成为科学焦点。它们的功能尚未完全理解，而一两种金属的存在是功能性，结构性还是共催化性。这项工作将在K99的资金期间开始，并将在独立阶段继续进行，并将尝试研究这些酶并绘制其功能的分子背景（水解与氧合）。该项目第二阶段的远距离目的是基于生物信息学，晶体学，诱变和活动研究的确定者在HD超家族中指导不同功能的决定者最终导致鉴定新的抗病毒药物因子和新的抗病毒药物因子和 治疗剂以及在化学困难的小分子转化中实现的新型氧合酶的发现。