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.

Public Health Relevance

Iron-containing enzymes are involved in many critical biosynthetic pathways, both in human cells and microbial pathogens. Interestingly, many of these enzymes are capable of effecting different reactions using the same active site, though the molecular details of how this is accomplished remain unknown. This project seeks a better understanding of how these enzymes conduct such transformations, which will inform the design of drugs to target these proteins and the development of novel chemical processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM113389-02
Application #
9014423
Study Section
Special Emphasis Panel (ZRG1-F04B-D (20))
Program Officer
Lees, Robert G
Project Start
2015-02-01
Project End
2018-01-31
Budget Start
2016-02-01
Budget End
2017-01-31
Support Year
2
Fiscal Year
2016
Total Cost
$54,294
Indirect Cost
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Fuller, Franklin D; Gul, Sheraz; Chatterjee, Ruchira et al. (2017) Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nat Methods 14:443-449
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
Martinie, Ryan J; Blaesi, Elizabeth J; Krebs, Carsten et al. (2017) Evidence for a Di-?-oxo Diamond Core in the Mn(IV)/Fe(IV) Activation Intermediate of Ribonucleotide Reductase from Chlamydia trachomatis. J Am Chem Soc 139:1950-1957
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
Rees, Julian A; Wandzilak, Aleksandra; Maganas, Dimitrios et al. (2016) Experimental and theoretical correlations between vanadium K-edge X-ray absorption and K? emission spectra. J Biol Inorg Chem 21:793-805
Pollock, Christopher J; DeBeer, Serena (2015) Insights into the geometric and electronic structure of transition metal centers from valence-to-core X-ray emission spectroscopy. Acc Chem Res 48:2967-75