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.
Iron- and 2-(oxo)glutarate-dependent oxygenase enzymes from plants, fungi, and soil bacteria carry out a bewildering array of oxidative transformations in biochemical pathways to many important natural-product drugs. Most of the reactions are not in the repertoire of synthetic chemists. The ability to reprogram their outcomes at will would enable creation of new drugs in a combinatorial way. This project will afford both an atom-level understanding of the principles of reaction control in these enzymes and specific variant enzymes capable of new, unnatural outcomes. Ultimately, tools for catalyst and natural-product drug design will emerge.
|Dunham, Noah P; Chang, Wei-Chen; Mitchell, Andrew J et al. (2018) Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of ?-Heteroatom Assistance. J Am Chem Soc 140:7116-7126|
|Martinie, Ryan J; Pollock, Christopher J; Matthews, Megan L et al. (2017) Vanadyl as a Stable Structural Mimic of Reactive Ferryl Intermediates in Mononuclear Nonheme-Iron Enzymes. Inorg Chem 56:13382-13389|
|Mitchell, Andrew J; Dunham, Noah P; Martinie, Ryan J et al. (2017) Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase. J Am Chem Soc 139:13830-13836|