Mechanistic Determination of the Non-heme Iron Enzyme Hyoscyamine-6-hydroxylase

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 DESCRIPTION (provided by applicant): Enzymes that employ non-heme-iron cofactors are involved in many important cellular processes, including transcription, reproduction, and the production of small molecule metabolites. One class of these enzymes, the iron- and 2-oxoglutarate (Fe/2OG) oxygenases, are notable in that some members can catalyze multiple different reactions using the same active site. Several of these reactivities (e.g. cyclizations, desaturations) cannot be understood by adapting the known mechanisms of these enzymes, motivating a deeper understanding of the chemistry involved. The enzyme hyoscyamine-6- hydroxylase (H6H) is a member of the Fe/2OG family and is responsible for the conversion of hyoscyamine to scopolamine, a tropane alkaloid pharmaceutical used in the treatment of conditions such as motion sickness and Parkinson's disease. This enzyme first hydroxylates hyoscyamine and then performs a subsequent dehydrogenation on the nascent alcohol to generate an epoxide ring. This second step is quite unusual in biology and its mechanism is not understood. In the proposed project, structures and mechanisms H6H will be determined with the goal of elucidating how this enzyme conducts these divergent reactions. X-ray crystallography will provide structural insights into how these reaction are mediated, while stopped-flow kinetics measurements will determine-in molecular detail-the precise mechanisms of both transformations. Furthermore, spectroscopic studies on the reaction intermediates will allow for definitive characterization of the chemical species involved in these reactions. Taken together, this work will help to rationalize the factors that govern the divergent reactivity of thse enzymes and will serve as a basis for the design of new medicinal compounds that target these enzymes.