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 在反应中未活化的脂肪族碳中心，这对 中心生命过程（例如，代谢及其调控，转录，表观遗传 遗传），并与多种疾病有关。植物、真菌和细菌已经多样化 Fe/2OG-加氧酶平台用于一系列令人困惑的氧化转化， 包括脂肪族化合物卤化、环化、双取代和立体转化 碳中心作为生物合成机器， 天然产物药物充满了这样的Fe/2OG加氧酶，重新编程的能力， 通过理性或定向进化的方法，它们将使微生物/酶 新药物化合物的自动生产。在这个项目中，我们将使用在体内和在 体外选择方法来工程化Fe/2OG加氧酶的结果改变的变体， 抗生素和麻醉药的途径。我们经过广泛验证的动力学和 光谱方法的结构和功能分析这些酶将是 结合两种新的、创新的结构方法， 在精确的结构和机制方面重新编程。该项目将提供 既有用于生产新的潜在药物的酶， 这些和其他Fe/2OG加氧酶的更合理的重编程。

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.