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

项目摘要/摘要 铁和2-氧戊二酸依赖(Fe/2OG)酶，代表非血红素单核超家族 含铁(NHM-Fe)酶已引起基础酶的强烈研究兴趣 近年来生物工程/生物催化探索的机理研究 催化反应性和简单的酶结构。C-H键活化的自由基卤化反应 Fe/2OG卤代酶催化在化学合成和生物催化应用中特别有吸引力， 由于这些酶可以以区域和立体特定的方式安装碳卤键，这一壮举迄今尚未 将通过有机合成方法学来实现。载体蛋白的机理研究揭示了- 依赖Fe/2OG卤代酶的自由基卤化机制的关键步骤是选择性卤化物 关键反应生成的羟基-Fe(III)-卤化物中间体向底物自由基的转移 物种，铁基(Fe(IV)=O)中间体。然而，一种解释选择性卤化物的共识机制 Fe/2OG卤代酶中的转移还没有达到，特别是避免羟基的控制因素 自由基转移导致的羟基化反应还没有完全揭示出来。此外，Fe/20OG的原因 酶不能进行氟化反应是完全未知的。在这个项目中，我们将架起这些桥梁 通过研究两种新发现的不依赖载体蛋白的Fe/2OG卤代酶来实现知识差距 催化氯化反应生成卤代核苷酸天然产物和卤化游离态 常备氨基酸。通过使用由机械探头设计和综合组成的一体化方法， 通过LC-MS和核磁共振分析确定酶产物结构，瞬时酶动力学，高级 光谱表征和分子动力学模拟，我们将阐明蛋白质- 底物相互作用和动力学在控制有效卤化中的作用，探索不同铁结合的影响 阴离子(例如，氯离子和氟离子)对铁基中间体的电子结构和反应性的影响，测试新的化学物质 使Fe/2OG酶氟化的策略，并扩大这些酶的底物范围 潜在的合成应用。考虑到含卤素的有机分子在现代社会中的重要性 这些新发现的卤代酶的机理阐明及其在药物和农用化学品中的应用 将为这些独特的酶未来的生物催化应用奠定科学基础。

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.