Mechanisms of Mononuclear non-Heme-Iron Enzymes

项目概要/摘要 利用含铁辅因子进行活性的酶催化一系列令人困惑的（通常非常 困难的）化学反应，这些化学反应对中心生命过程至关重要（例如，DNA生物合成 修复，基因调控，表观遗传的调控，大量化合物的生物合成， 抗细菌和抗真菌活性）。这些酶的功能障碍通常与 严重疾病，如癌症、心血管疾病和糖尿病。利用合成药物的战略 这些酶的潜力和对抗与其功能障碍相关的疾病涉及合理的 在分子水平上操纵这些过程。这一奋进的先决条件是详细了解 基本的反应机制，特别是酶如何控制其反应的结果。 Bollinger/Krebs联合小组专门从事将瞬态快速动力学实验与各种 光谱（例如停流吸收、冷冻淬灭EPR和穆斯堡尔谱）和分析（LC/MS） 监测金属酶反应的方法。在过去的15年里，他们的团队成功地研究了许多 - 需要单核或双核非血红素铁辅因子通过捕获来进行活性的酶， 表征其催化循环中的关键反应中间体。特别是，他们发现了高自旋Fe（IV）- 几种单核非血红素铁酶中的氧代（铁基）中间体，主要是Fe（II）-和2-氧代-戊二酸- 依赖性（Fe/2 OG）酶。铁基中间体通常通过切割铁基中间体来引发底物氧化。 脂肪族C-H键。这些反应的结果是多种多样的，包括羟基化（默认 产物）、卤化、去饱和、差向异构化和杂环化反应。许多反应 用于重要医学天然产物的生物合成。目前的研究重点是 Bollinger/Krebs小组旨在破译导致不同结果的因素。的长期目标 本研究旨在为生物技术中酶的合理操作奠定基础。 应用. PI还与Squire Booker就Fe/S的机理研究进行了长期合作 酶，特别是属于自由基S-腺苷甲硫氨酸（RS）酶超家族的那些。 这些酶使用[4Fe-4S]簇来产生典型的5 '-脱氧-腺苷-5'-基自由基，其引发 各种各样的底物氧化，通常通过脂肪族C-H键的断裂。的当前焦点 对RS酶的合作研究旨在阐明不同反应的反应机理 结果，即硫插入、甲基化、甲基硫醇化和去饱和。

Project Summary/Abstract Enzymes that utilize iron-containing cofactors for their activity catalyze a bewildering array of (often very difficult) chemical reactions that are fundamentally important to central life processes (e.g., DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of a plethora of 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 combat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level. A prerequisite for this endeavor is a detailed knowledge of the underlying 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 15 years, their group has successfully studied many enzymes that require a mononuclear or a dinuclear non-heme-iron cofactor for activity by trapping and characterizing key reaction intermediates in their catalytic cycles. In particular, they identified high-spin Fe(IV)- oxo (ferryl) intermediates in several mononuclear non-heme-iron enzymes, mostly Fe(II)- and 2-oxo-glutarate- dependent (Fe/2OG) 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, and heterocyclization reactions. Many of these reactions are employed in the biosyntheses of medically important natural products. The current focus of research in the Bollinger/Krebs group 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. The PI also has a long-standing collaboration with Squire Booker on mechanistic studies of Fe/S enzymes, in particular those that belong to the superfamily of radical S-adenosylmethionine (RS) enzymes. These enzymes use a [4Fe-4S] cluster to generate a canonical 5’-deoxy-adenos-5’-yl radical that initiates a wide variety of substrate oxidations, often by cleavage of an aliphatic C-H bond. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of different reaction outcomes, viz sulfur insertion, methylation, methylthiolation, and desaturation.