Tuesday, January 14, 2025
1/14/2025
Summary / Abstract Radical SAM is the largest enzyme superfamily known, with its members catalyzing a remarkable diversity of reactions in all domains of life. Radical SAM enzymes are involved in the synthesis of essential cofactors and antibiotics, repair of DNA damage, and the assembly of complex biological metal clusters, among many other reactions. The presence of radical SAM enzymes in humans as well as in both beneficial and pathogenic microbes lends high significance to understanding their fundamental properties and mechanisms. The proposed research will develop a critical understanding of radical SAM mechanisms, including both the radical initiation process common to all enzymes in this large and diverse superfamily, and mechanisms of individual radical SAM enzymes. Research efforts will focus on freeze-quench trapping radical intermediates and using time-delay, thermal annealing, and photo-initiation to probe reaction steps and chemical properties. Trapped intermediates will be characterized by using spectroscopic approaches such as electron paramagnetic resonance and electron-nuclear double resonance, and structural approaches such as X-ray crystallography. Systems to be studied will include the pyruvate formate-lyase activating enzyme, which has proven to be an outstanding prototypical radical SAM enzyme for illuminating mechanistic details. In addition, we will pursue mechanistic insights into radical SAM enzymes involved in maturation of RiPPs (ribosomally-encoded, post-translationally modified peptides) and larger protein substrates undergoing substantial modification. We will further be working to define the biochemical function of RSAD1, a radical SAM enzyme implicated in Alzheimer’s disease in humans. This project will provide new insights into the fundamental mechanisms of radical initiation in radical SAM enzymes, and into the mechanistic and functional details of some of the more complex and interesting reactions catalyzed by members of this enzyme superfamily.
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