Dissect Mechanism of Iron(II)/2-Oxoglutarate Dependent Enzymes Catalyzed Halogenation in Nucleotide Biosynthesis

核苷酸生物合成中铁(II)/2-氧化戊二酸依赖性酶催化卤化的解析机制

• 批准号: 10660003
• 负责人: Yisong Guo
• 金额: $ 35.51万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660003
• 关键词: 2' -deoxyadenosine; Agrochemicals; Alkenes; Alkynes; Amino Acids; Anabolism; Anions; Antineoplastic Agents; Architecture; Azides; Binding; Biomedical Engineering; Carbon; Carrier Proteins; Chemicals; Chlorides; Consensus; Copper; Data; Electronics; Environment; Enzyme Kinetics; Enzymes; Exhibits; Family; Foundations; Future; Grant; Halogens; Homologous Gene; Hydrogen; Hydrogen Bonding; Hydroxyl Radical; Hydroxylation; Iron; Knowledge; Leucine; Libraries; Methodology; Modernization; Mononuclear; Mutagenesis; Natural Products; Nucleosides; Nucleotide Biosynthesis; Nucleotides; Outcome; Oxidants; Oxygen; Pathway interactions; Pharmacologic Substance; Positioning Attribute; Property; Proteins; Quinolones; Race; Reaction; Research; Science; Shapes; Site; Substrate Interaction; Testing; Viral; alpha ketoglutarate; chemical synthesis; chlorination; cycloaddition; design; drug discovery; electronic structure; enzyme mechanism; enzyme substrate; fluorophore; geometric structure; halogenation; insight; interest; molecular dynamics; novel; screening; spectroscopic survey; synthetic drug

Project Summary/Abstract Iron and 2-oxoglutarate-dependent (Fe/2OG) enzymes, representing a superfamily of non-heme mononuclear iron-containing (NHM-Fe) enzymes, have garnered strong research interests from fundamental enzyme mechanism studies to bioengineering/biocatalysis explorations in recent years due to their exceedingly diverse catalytic reactivities and simple enzyme architectures. Radical halogenation reactions via C-H bond activation catalyzed by Fe/2OG halogenases are particularly attractive for chemical synthesis and biocatalysis applications, since these enzymes can install carbon-halide bonds in a regio- and stereo-specific manner, a feat that has yet to be achieved by organic synthetic methodology. As revealed by the mechanistic studies of carrier protein- dependent Fe/2OG halogenases, the key step in the radical halogenation mechanism is the selective halide radical transfer from the hydroxo-Fe(III)-halide intermediate to the substrate radical generated by the key reactive species, the ferryl (Fe(IV)=O) intermediate. However, a consensus mechanism to explain the selective halide transfer in Fe/2OG halogenases has not been reached, particularly the controlling factors to avoid hydroxyl radical transfer to lead to hydroxylation reaction are not fully revealed. Additionally, the reasons why Fe/2OG enzymes cannot perform fluorination reaction are completely unknown. In this project, we will bridge these knowledge gaps by studying two newly discovered carrier protein-independent Fe/2OG halogenases that catalyze chlorination reactions to generate halogenated nucleotide natural products and halogenated free- standing amino acids. By using an integrative approach consisting of mechanistic probe design and synthesis, enzyme product structural determination via LC-MS and NMR analysis, transient enzyme kinetics, advanced spectroscopic characterization and molecular dynamics simulations, we will elucidate the influence of protein- substrate interactions and dynamics in controlling efficient halogenation, explore the effect of different iron-bound anions (e.g. Cl- vs. F-) to the electronic structure and the reactivity of the ferryl intermediate, test new chemical strategies to enable fluorination in Fe/2OG enzymes, and expand the substrate scope of these enzymes for potential synthetic applications. Given the importance of halogen-containing organic molecules in the modern pharmaceutical and agrochemical applications, mechanistic elucidation of these newly discovered halogenases will lay scientific foundation for future biocatalytic applications of these unique enzymes.

项目概要/摘要 铁和 2-酮戊二酸依赖性 (Fe/2OG) 酶，代表非血红素单核超家族 含铁（NHM-Fe）酶，引起了基础酶的强烈研究兴趣 近年来生物工程/生物催化探索的机制研究因其极其多样化 催化反应性和简单的酶结构。通过 C-H 键活化的自由基卤化反应 由 Fe/2OG 卤化酶催化对于化学合成和生物催化应用特别有吸引力， 由于这些酶可以以区域和立体特异性的方式安装碳卤化物键，这一壮举尚未实现 通过有机合成方法实现。正如载体蛋白的机制研究所揭示的那样—— 依赖Fe/2OG卤化酶，自由基卤化机制的关键步骤是选择性卤化物 从羟基-Fe(III)-卤化物中间体到由关键反应生成的底物自由基的自由基转移 种，ferryl (Fe(IV)=O) 中间体。然而，解释选择性卤化物的共识机制 Fe/2OG 卤化酶的转移尚未实现，特别是避免羟基的控制因素 自由基转移导致羟基化反应尚未完全揭示。另外，Fe/2OG的原因 酶不能进行氟化反应是完全未知的。在这个项目中，我们将桥接这些 通过研究两种新发现的不依赖于载体蛋白的 Fe/2OG 卤化酶来弥补知识空白 催化氯化反应生成卤代核苷酸天然产物和卤代游离产物 固定氨基酸。通过使用由机械探针设计和合成组成的综合方法， 通过 LC-MS 和 NMR 分析、瞬时酶动力学、高级酶产物结构测定 光谱表征和分子动力学模拟，我们将阐明蛋白质的影响 底物相互作用和动力学在控制有效卤化中的作用，探索不同铁结合的效果 阴离子（例如 Cl- 与 F-）对 Ferryl 中间体的电子结构和反应性的影响，测试新化学品 使 Fe/2OG 酶氟化并扩大这些酶的底物范围的策略 潜在的合成应用。鉴于含卤素有机分子在现代工业中的重要性 药物和农业化学应用，这些新发现的卤化酶的机理阐明 将为这些独特酶的未来生物催化应用奠定科学基础。

项目成果

Enabling Valence Delocalization in Iron(III) Macrocyclic Complexes through Ring Unsaturation.

• DOI: 10.1021/acs.inorgchem.3c01179
• 发表时间: 2023-06
• 期刊: Inorganic chemistry
• 影响因子: 4.6
• 作者: Reese A. Clendening;Stephanie S Delancey;Andrew T Poore;Shan Xue;Yisong Guo;Shiliang Tian;T. Ren

Harnessing the Substrate Promiscuity of Dioxygenase AsqJ and Developing Efficient Chemoenzymatic Synthesis for Quinolones.

• DOI: 10.1021/acscatal.1c01150
• 发表时间: 2021-06-18
• 期刊: ACS CATALYSIS
• 影响因子: 12.9
• 作者: Tang, Haoyu;Tang, Yijie;Kurnikov, Igor, V;Liao, Hsuan-Jen;Chan, Nei-Li;Kurnikova, Maria G.;Guo, Yisong;Chang, Wei-chen
• 通讯作者: Chang, Wei-chen