ConspectusThe seeds for recognition of the vast superfamily of radical S-adenosyl-l-methionine (SAM) enzymes were sown in the 1960s, when Joachim Knappe found that the dissimilation of pyruvate was dependent on SAM and Fe(II), and Barker and co-workers made similar observations for lysine 2,3-aminomutase. These intriguing observations, coupled with the evidence that SAM and Fe were cofactors in radical catalysis by these enzyme systems, drew us in the 1990s to explore how Fe(II) and SAM initiate radical reactions. Our early work focused on the same enzyme Knappe had originally characterized: the pyruvate formate-lyase activating enzyme (PFL-AE). Our discovery of an iron-sulfur cluster in this enzyme, together with similar findings for other SAM-dependent enzymes at the time, led to the recognition of an emerging class of enzymes that use iron-sulfur clusters to cleave SAM, liberating the 5′-deoxyadenosyl radical (5′-dAdo•) that initiates radical reactions. A major bioinformatics study by Heidi Sofia and co-workers identified the enzyme superfamily denoted Radical SAM, now known to span all kingdoms of life with more than 100,000 unique sequences encoding enzymes that catalyze remarkably diverse reactions.Despite the limited sequence similarity and vastly divergent reactions catalyzed, the radical SAM enzymes appear to employ a common mechanism for initiation of radical chemistry, a mechanism we have helped to clarify over the last 25 years. A reduced [4Fe-4S]+ cluster provides the electron needed for the reductive cleavage of SAM. The resulting [4Fe-4S]2+ cluster can be rereduced either by an external reductant, with SAM acting as a cosubstrate, or by an electron provided during the reformation of SAM in cases where SAM is used as a cofactor. The amino and carboxylate groups of SAM bind to the unique iron of the catalytic [4Fe-4S] cluster, placing the sulfonium of SAM in close proximity to the cluster. Surprising recent results have shown that the initiating enzymatic cleavage of SAM generates an organometallic intermediate prior to liberation of 5′-dAdo•, which initiates radical chemistry on the substrate. This organometallic intermediate, denoted ω, has a 5′-deoxyadenosyl moiety directly bound to the unique iron of the [4Fe-4S] cluster via the 5′-C, giving a structure that is directly analogous to the Co-(5′-C) bond of the organometallic cofactor adenosylcobalamin. Our observation that this intermediate ω is formed throughout the superfamily suggests that this is a key intermediate in initiating radical SAM reactions, and that organometallic chemistry is much more broadly relevant in biology than previously thought.
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