This proposal is focused on exploring the fundamental chemical mechanism of a large superfamily of enzymes called radical SAM enzymes. These enzymes occur in humans in such diverse functions as cofactor synthesis and the antiviral response, and are found in nature in all kingdoms of life. The radical SAM superfamily is large, with thousands of identified members catalyzing a range of reactions;they have in common the utilization of a specific type of metal cluster (a [4Fe-4S] cluster) and S-adenosyl-L-methionine (SAM) to initiate radical chemistry on their substrates. The current proposal will focus primarily on three members of the radical SAM superfamily (pyruvate formate-lyase activating enzyme, lysine 2,3-aminomutase, and viperin) that have diverse functions, and experiments will range from fundamental mechanistic, spectroscopic, and structural studies aimed at delineating details of the chemical mechanisms to basic functional studies aimed at understanding the role of an important mammalian radical SAM enzyme.
The specific aims of the proposal are: 1) To identify specific steps in the catalytic mechanisms of these enzymes by using techniques designed to trap reaction intermediates, 2) To explore valence localization in the [4Fe-4S] cluster of these enzymes, its causes, and its implications for function, 3) To structurally characterize the interaction between one radical SAM enzyme and its protein substrate, and 4) to functionally characterize a human radical SAM enzyme involved in the antiviral response.

Public Health Relevance

The proposed work will provide fundamental new insights into a class of enzymes that are widespread in nature. These enzymes participate in numerous processes relevant to public health, including the response of the human body to viral infections and the synthesis of essential vitamins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Montana State University - Bozeman
Schools of Arts and Sciences
United States
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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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