Radical <i>S</i>-adenosyl-L-methionine (SAM) enzymes are ubiquitous and diverse, catalyzing radical reactions in key pathways throughout all kingdoms of life. Although the ultimate reactions are disparate, the initiation mechanism involving iron-sulfur cluster-mediated reductive cleavage of SAM to produce a 5’-deoxyadenosyl radical intermediate is thought to be a common feature in all radical SAM enzymes. In order to probe the stepwise mechanism of this radical initiation process, we have used chemical and spectroscopic methods to trap and characterize intermediate states. Use of the SAM analog <i>S</i>-3’,4’-anhydroadenosyl-L-methionine (anSAM) allows production of the allylically stabilized anhydroadenosyl radical intermediate. We have used electron-nuclear double resonance spectroscopy of this stabilized radical intermediate in the radical SAM enzyme lysine 2,3-aminomutase to reveal how the active site exerts van der Waals control of the reacting radical, allowing only the most minimal movements as the anhydroadenosyl radical is guided to the substrate hydrogen. In this way, lysine aminomutase tames the 5’-deoxyadenosyl radical, preventing it from carrying out harmful side reactions. As a complement to using the anSAM analog, we have examined the reaction of SAM with the enzyme pyruvate formate-lyase activating enzyme (PFL-AE) using rapid freeze-quench coupled to electron paramagnetic resonance and electron-nuclear double resonance spectroscopies. Our results reveal formation of a catalytically competent intermediate in which the 5’-C of a 5’-deoxyadenosyl moiety is directly bonded to the unique iron of the [4Fe-4S] cluster of PFL-AE. The surprising discovery of this organometallic intermediate leads to fundamental questions regarding the mechanism by which the intermediate forms, the mechanistic purpose of this intermediate, and whether this intermediate is common to all radical SAM enzymes.

Mechanism and control in radical SAM enzymes

253rd National Meeting (2017)

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