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

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结构域中有超过37，000个成员。虽然有一般注释为（磷酸）水解酶，其功能大多是未知的。不到十年前，一种新的二铁HD酶参与肌醇的催化并与I型糖尿病相关，即肌醇加氧酶，被证明使用分子氧进行完全不同的反应以提供其底物的活化。最近唯一鉴定的HD酶PhnZ也显示出遵循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。活性中间体的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的机制惊人的相似性，引发了关于这些酶的功能及其对人类健康和环境的可能影响的问题。为此，该项目的第一部分将侧重于PhnZ的表征。这需要结合光谱，结构，氧化还原和活性研究，以建立这种新的加氧酶的工作方式。将研究酶的无底物和结合形式，以便首次获得关于酶的结构和电子信息。 “开”和“关”的反应状态，其中晶体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奠定基础，从而对它们的功能进行有力的控制。此外，化学反应中下游事件的表征将可能建立特定非血红素铁酶（单核或双核）采取的共同策略，以进行困难和对环境重要的反应。该项目的第二部分旨在绘制（双核）HD结构域酶的催化景观，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，已经确定了新的有吸引力的未知功能的蛋白质靶标。选择的蛋白质将被过表达和纯化。这些将结合EPR、穆斯堡尔谱、晶体学和NMR技术进行光谱表征。将通过筛选特定底物的活性和质谱法确定其活性概况。目前，有少数HD结构域酶参与免疫应答，如HIV-1的限制因子或攻击病毒核苷酸的核苷酸酶已成为科学焦点。它们的功能尚未完全了解，而一种或两种金属的存在是不知道它是功能性的，结构性的还是共催化性的。这项工作将在K99资助期间开始，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（水解与氧化）。该项目第二阶段的长期目的是在生物信息学、晶体学、诱变和活性研究的基础上建立指导HD超家族内不同功能的决定因素，这将最终导致鉴定新的抗病毒因子， 治疗剂以及涉及化学上困难的小分子转化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.