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

描述(申请人提供)：HD结构域蛋白构成了一个新的金属酶超家族，目前在所有三个生命领域都有超过37,000个成员。虽然通常被注释为AS(磷酸)水解酶，但它们的功能大多不清楚。不到十年前，一种参与肌醇分解代谢并与I型糖尿病相关的新型双铁HD酶，即肌醇加氧酶，被证明利用分子氧来进行完全不同的反应，以提供其底物的激活。最近发现的唯一HD酶PhnZ也被证明遵循MIOX的范例，海洋微生物利用氧气将有机磷酸盐转化为磷酸盐。这些反应中间体的作用机制和结构目前尚不清楚，但它们与参与抗生素生物合成的其他非血红素铁酶有着惊人的相似性，这引发了人们对这些酶的功能及其对人类健康和环境的可能影响的疑问。为此，该项目的第一部分将侧重于PhnZ的特征。这需要结合光谱、结构、氧化还原和活性研究，以建立这种新型加氧酶的工作方式。将研究该酶的无底物和结合形式，以便首次获得关于该酶的结构和电子信息。 ‘开’和‘关’的反应状态的结晶学信息一直是极具挑战性的，底物或抑制剂如何调整和影响酶活性部位的性质。这些信息将为发现能够激活和抑制这些酶的化合物奠定基础，从而提供对其功能的强大控制。此外，化学反应中下游事件的表征可能会建立特定的非血红素铁酶(单核或双核)进行困难和对环境重要的反应所采用的共同策略。该项目的第二部分旨在绘制(双核)HD结构域酶的催化图谱，发现新的功能，并确定金属在调节特定活动(水解与氧化)中的类型和作用。为此，在系统发育分析的基础上，新的具有吸引力的功能未知的蛋白质靶点已经被确定。选定的蛋白质将被过度表达和纯化。这些化合物将结合电子顺磁共振、M�穆斯堡尔谱、结晶学和核磁共振技术进行光谱表征。通过筛选特定底物的活动和质谱学方法，将建立它们的活动概况。目前，有几种HD结构域的酶与免疫反应有关，如HIV-1的限制因子或攻击病毒核苷酸的核苷酸酶，这些都已成为科学关注的焦点。它们的功能还不完全清楚，而一种或两种金属的存在也不知道它是功能的、结构的还是共催化的。这项工作将在K99资助期开始，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(水解性与氧合性)。该项目第二阶段的长期目标是在生物信息学、结晶学、诱变和活性研究的基础上建立决定因素，这些决定因素将指导HD超家族的不同功能，最终将导致识别新的抗病毒因子和 治疗药物以及与化学上困难的小分子转化有关的新型加氧酶的发现。

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.