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

人铁(II)和2-(氧代)戊二酸依赖(Fe/2OG)双加氧酶羟化 反应中未活化的脂肪族碳中心对 中心生命过程(如新陈代谢及其调控、转录、表观遗传 遗传)，并与几种疾病有关。植物、真菌和细菌已经多样化 一系列令人眼花缭乱的氧化转化的Fe/20G加氧酶平台 包括卤化、环化、脱氢和脂肪族立体转化 碳中心。由于生物合成机械产生了大量重要的 天然产物药物充满了这样的Fe/2OG加氧酶，即重新编程的能力 通过理性或定向进化的方法将使微生物/ENZY- 马季奇制作的新型药物化合物。在这个项目中，我们将使用体内和体内 铁/2OG加氧酶突变体的体外筛选方法 抗生素和麻醉剂药物的途径。我们经过广泛验证的动能和 这些酶的结构和功能分析的光谱方法将是 与两种新的、创新的结构方法相结合应用，使 以精确的结构和机械术语重新编程。因此，该项目将提供 既有用于生产新的潜在药物的酶，也有使 对这些和其他Fe/2OG加氧酶进行更合理的重新编程。

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.