Mechanisms and Reprogramming of Iron/2-Oxoglutarate Desaturases and Oxacyclases

人铁 (II)- 和 2-(氧代) 戊二酸依赖性 (Fe/2OG) 双加氧酶羟基化 反应中未活化的脂肪族碳中心对于 中心生命过程（例如新陈代谢及其调节、转录、表观遗传 遗传）并与多种疾病相关。植物、真菌和细菌已经多样化 Fe/2OG 加氧酶平台可进行一系列令人眼花缭乱的氧化转化 包括脂肪族的卤化、环化、脱氢和立体反转 碳中心。随着生物合成机器产生大量重要的 天然产物药物富含这种 Fe/2OG 加氧酶，具有重新编程的能力 通过理性或定向进化方法，它们将使微生物/酶 新型药物化合物的自动化生产。在这个项目中，我们将使用 in vivo 和 in 体外选择方法来设计Fe/2OG加氧酶的结果改变变体 抗生素和麻醉药物的途径。我们经过广泛验证的动力学和 对这些酶进行结构和功能分析的光谱方法将 结合两种新的、创新的结构方法来合理化 以精确的结构和机械术语重新编程。因此，该项目将提供 用于生产新的潜在药物的酶和机制洞察力使 对这些和其他 Fe/2OG 加氧酶进行更合理的重编程。

Human iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) dioxygenases hydroxylate unactivated aliphatic carbon centers in reactions that are fundamentally important to central life processes (e.g., metabolism and its regulation, transcription, epigenetic inheritance) and relevant to several diseases. Plant, fungi, and bacteria have diversified the Fe/2OG-oxygenase platform for a bewildering array of oxidative transformations that include halogenations, cyclizations, dehydrogenations and stereoinversions of aliphatic carbon centers. As the biosynthetic machinery generating a large number of important natural-product drugs are replete with such Fe/2OG oxygenases, the ability to reprogram them by either rational or directed-evolution approaches would enable microbial/enzy- matic production of novel drug compounds. In this project, we will use in vivo and in vitro selection methods to engineer outcome-altered variants of Fe/2OG oxygenases on pathways to antibiotic and anesthetic drugs. Our extensively validated kinetic and spectroscopic approaches to structural and functional analysis of these enzymes will be applied in combination with two new, innovative structural methods to rationalize the reprogramming in precise structural and mechanistic terms. The project will thus provide both enzymes for production of new potential drugs and mechanistic insight to enable more rational reprogramming of these and other Fe/2OG oxygenases.