Mechanisms of Iron-Containing Enzymes

项目摘要/摘要 利用含铁辅因子的酶催化许多不同的、通常非常困难的化学反应，这些反应 对核心生命过程至关重要，如DNA生物合成和修复、基因调控、 多种抗菌、抗真菌化合物的表观遗传、生物合成调控 活动。这些酶的功能障碍通常与严重疾病的发生有关，例如癌症， 心血管疾病和糖尿病。利用这些酶的合成潜力并 治疗与其功能障碍相关的疾病涉及对这些过程的合理操纵 分子水平，这需要详细的反应机理知识，特别是酶是如何 控制他们反应的结果。布林格/克雷布斯联合小组专门研究组合暂态 不同光谱的快速动力学实验(例如停流吸收、冷冻-淬火EPR和 Mössbauer)和分析(LC/MS)方法来监测金属酶反应。在过去的20年里，我们集团 通过捕获成功地研究了许多单核(MNHI)和双核非血红素铁(DNHI)酶 以及表征催化循环中的关键反应中间体。我们捕获并描述了高自旋 各种MNHI酶中的Fe(IV)-氧代(铁基)中间体。铁基中间体引发底物氧化， 通常是通过裂解脂肪族的C-H键。这些反应的结果是不同的，包括 羟基化(默认结果)、卤化、脱饱和、异构化、杂环化和 内过氧化反应。我们目前的研究重点是破译导致这种现象的因素。 结果多种多样。这项研究的长期目标是为合理操作 这些酶用于生物技术应用。与Boal小组合作，B/K小组有 鉴定了血红素加氧酶二铁加氧酶(HDO)酶超家族，目前研究了几个 HDOS。国际和平协会还与布克小组就激进分子S的机械研究进行了长期的合作- 腺苷蛋氨酸(RS)酶，催化形成新的C-S键。这些酶使用它们的[Fe4S4]RS 生成典型的5‘-脱氧腺苷-5’-基自由基，该自由基通过切割底物引发底物氧化 脂肪族C-H键及其辅助的[Fe4S4]团簇作为硫源。当前协作的重点是 对RS酶的研究旨在阐明各种RS酶参与的反应机制。 形成新的C-S键。

Project Summary/Abstract Enzymes that utilize iron-containing cofactors catalyze many different, often very difficult, chemical reactions that are fundamentally important to central life processes, such as DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of numerous compounds with antibacterial and antifungal activities. Dysfunction of these enzymes is often associated with the onset of severe diseases, e.g. cancer, cardiovascular diseases, and diabetes. Strategies to harness the synthetic potential of these enzymes and to treat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level, which requires a detailed knowledge of the reaction mechanisms, in particular how the enzymes control the outcome of their reactions. The Bollinger/Krebs joint group specializes in combining transient-state rapid kinetic experiments with various spectroscopic (e.g. stopped-flow absorption, freeze-quench EPR and Mössbauer) and analytical (LC/MS) methods to monitor metalloenzyme reactions. In the last 20 years, our group has successfully studied many mononuclear (MNHI) and dinuclear non-heme-iron (DNHI) enzymes by trapping and characterizing key reaction intermediates in their catalytic cycles. We trapped and characterized high-spin Fe(IV)-oxo (ferryl) intermediates in various MNHI enzymes. The ferryl intermediate initiates substrate oxidation, typically by cleavage of an aliphatic C-H bond. The outcome of these reactions is diverse and includes hydroxylation (the default outcome), halogenation, desaturation, epimerization, heterocyclization, and endoperoxidation reactions. The current focus of our research aims at deciphering the factors that result in the diverse outcomes. The long-term goal of this research is to lay the foundation for the rational manipulation of these enzymes for biotechnological applications. In collaboration with the Boal group, the B/K group has identified the heme-oxygenase diiron oxygenase (HDO) enzyme superfamily and currently studies several HDOs. The PI also has a long-standing collaboration with the Booker group on mechanistic studies of radical S- adenosylmethionine (RS) enzymes that catalyze formation of new C-S bonds. These enzymes use their [Fe4S4]RS cluster to generate the canonical 5’-deoxy-adenos-5’-yl radical that initiates substrate oxidation by cleavage of aliphatic C-H bonds and their auxiliary [Fe4S4] clusters as sulfur source. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of various RS enzymes involved in formation of new C-S bonds.