Mechanisms of Mononuclear non-Heme-Iron Enzymes

单核非血红素铁酶的机制

• 批准号: 9918429
• 负责人: CARSTEN KREBS
• 金额: $ 49.25万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918429
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotics; Antifungal Agents; Biotechnology; Cardiovascular Diseases; Chemicals; Collaborations; DNA Repair Gene; DNA biosynthesis; Diabetes Mellitus; Disease; Enzymes; Foundations; Freezing; Functional disorder; Gene Expression Regulation; Goals; Health; Human; Hydrogen Bonding; Hydroxylation; Iron; Joints; Kinetics; Knowledge; Life; Malignant Neoplasms; Medical; Methods; Methylation; Molecular; Monitor; Mononuclear; Natural Products; Outcome; Oxygen; Process; Reaction; Regulation; Research; S-Adenosylmethionine; Societies; Sulfur; absorption; alpha ketoglutarate; chemical reaction; cofactor; combat; epigenetic regulation; epimerization; experimental study; halogenation; liquid chromatography mass spectrometry; metalloenzyme; oxidation

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.

项目概要/摘要 利用含铁辅助因子发挥其活性的酶会催化一系列令人眼花缭乱的物质（通常非常 对中心生命过程（例如 DNA 生物合成）至关重要的化学反应 和修复、基因调控、表观遗传调控、大量化合物的生物合成 抗菌和抗真菌活性）这些酶的功能障碍通常与疾病的发生有关。 严重疾病，例如癌症、心血管疾病和糖尿病。 这些酶的潜力以及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的先决条件是详细的知识。 潜在的反应机制，特别是酶如何控制其反应的结果。 Bollinger/Krebs 联合小组专门致力于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）和分析（LC/MS） 监测金属酶反应的方法 在过去 15 年里，他们的小组成功地研究了许多方法。 需要单核或双核非血红素铁辅助因子通过捕获和 表征了催化循环中的关键中间反应，特别是，他们鉴定了高自旋 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- Ferrel 中间体通常通过裂解酶来启动底物氧化。 这些反应的结果多种多样，包括羟基化（默认）。 结果）、卤化、去饱和、差向异构化和杂环化反应中的许多反应。 用于医学上重要的天然产物的生物合成是当前研究的焦点。 Bollinger/Krebs 小组的目标是破译导致不同结果的因素。 本研究旨在为生物技术中合理操纵这些酶奠定基础 该 PI 还与 Squire Booker 在 Fe/S 机理研究方面有着长期合作。 酶，特别是那些属于自由基S-腺苷甲硫氨酸(RS)酶超家族的酶。 这些酶使用 [4Fe-4S] 簇生成典型的 5'-脱氧腺苷-5'-基自由基，从而引发 各种各样的底物氧化，通常是通过脂肪族 C-H 键的断裂来实现，这是当前的焦点。 RS酶的合作研究工作旨在描绘不同反应的反应机制 结果，即硫插入、甲基化、甲硫基化和去饱和。