The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (the so-called radical SAM) superfamily of enzymes. These enzymes span a remarkably diverse range of reactions and appear to be represented across the phylogenetic kingdom, with hundreds of radical SAM enzymes identified. The widespread occurrence of these enzymes throughout biology, from bacteria to humans, is indicative of the significance of the chemistry catalyzed by these enzymes. In humans, radical SAM enzymes are involved in the biosynthesis of lipoic acid, the synthesis of heme, and the biosynthesis of the molybdopterin cofactor, among many other essential functions, some as yet unidentified. Despite the diversity of reactions catalyzed, our overriding hypothesis is that the adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with S-adenosylmethionine to generate an adenosyl radical intermediate, which is directly involved in catalysis. These reactions represent novel chemistry for iron-sulfur clusters. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) will be pursued.
The specific aims i nclude further investigation of the [4Fe-4S] cluster of PFL-AE, its unique iron site, its interactions with substrate, and its role in catalysis. In addition we propose to explore the nature of the PFL-AE iron-sulfur cluster in whole cells. We will also utilize substrate analogs as probes of ES interactions and the PFL-AE catalytic mechanism. We will use spectroscopic approaches, particularly ENDOR, to probe the structural and electronic features of the PFL- AE/AdoMet/PFL complex. We will also utilize cryoreduction and rapid-freeze-quench spectroscopic approaches to identify and spectroscopically characterize intermediates in the radical-generation reaction catalyzed by PFL-AE. Finally, we will pursue structural characterization of PFL-AE and its complexes with PFL and SAM using X-ray crystallography.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054608-13
Application #
7622615
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Ikeda, Richard A
Project Start
1997-08-01
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2011-05-31
Support Year
13
Fiscal Year
2009
Total Cost
$249,095
Indirect Cost
Name
Montana State University - Bozeman
Department
Type
Schools of Arts and Sciences
DUNS #
625447982
City
Bozeman
State
MT
Country
United States
Zip Code
59717
Shepard, Eric M; Byer, Amanda S; Aggarwal, Priyanka et al. (2017) Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation. Biochemistry 56:3234-3247
Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki et al. (2017) Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme. J Am Chem Soc 139:11803-11813
Shepard, Eric M; Byer, Amanda S; Broderick, Joan B (2017) Iron-Sulfur Cluster States of the Hydrogenase Maturase HydF. Biochemistry 56:4733-4734
Horitani, Masaki; Shisler, Krista; Broderick, William E et al. (2016) Radical SAM catalysis via an organometallic intermediate with an Fe-[5'-C]-deoxyadenosyl bond. Science 352:822-5
Broderick, Joan B; Moody, James D (2016) Cutting Choline with Radical Scissors. Cell Chem Biol 23:1173-1174
Shepard, Eric M; Byer, Amanda S; Betz, Jeremiah N et al. (2016) A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF. Biochemistry 55:3514-27
Lill, Roland; Broderick, Joan B; Dean, Dennis R (2015) Special issue on iron-sulfur proteins: Structure, function, biogenesis and diseases. Biochim Biophys Acta 1853:1251-2
Horitani, Masaki; Byer, Amanda S; Shisler, Krista A et al. (2015) Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• ""Free Radical"" Is Never Free. J Am Chem Soc 137:7111-21
Broderick, Joan B; Duffus, Benjamin R; Duschene, Kaitlin S et al. (2014) Radical S-adenosylmethionine enzymes. Chem Rev 114:4229-317
Crain, Adam V; Broderick, Joan B (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J Biol Chem 289:5723-9

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