Radical SAM enzymes: Nature's choice for radical reactions

Abstract

Enzymes that use a [4Fe-4S]¹⁺ cluster plus S-adenosyl-l-methionine (SAM) to initiate radical reactions (radical SAM) form the largest enzyme superfamily, with over half a million members across the tree of life. This review summarizes recent work revealing the radical SAM reaction pathway, which ultimately liberates the 5'-deoxyadenosyl (5'-dAdo•) radical to perform extremely diverse, highly regio- and stereo-specific, transformations. Most surprising was the discovery of an organometallic intermediate Ω exhibiting an Fe-C5'-adenosyl bond. Ω liberates 5'-dAdo• through homolysis of the Fe-C5' bond, in analogy to Co-C5' bond homolysis in B₁₂ , previously viewed as biology's paradigmatic radical generator. The 5'-dAdo• has been trapped and characterized in radical SAM enzymes via a recently discovered photoreactivity of the [4Fe-4S]⁺ /SAM complex, and has been confirmed as a catalytically active intermediate in enzyme catalysis. The regioselective SAM S-C bond cleavage to produce 5'-dAdo• originates in the Jahn-Teller effect. The simplicity of SAM as a radical precursor, and the exquisite control of 5'-dAdo• reactivity in radical SAM enzymes, may be why radical SAM enzymes pervade the tree of life, while B₁₂ enzymes are only a few.

Keywords: B12; S-adenosylmethionine; adenosylcobalamin; deoxyadenosyl radical; electron paramagnetic resonance; mechanism; radical; radical SAM.

Figure 1.

Adenosylcobalamin, or coenzyme B12 (left) and the SAM-[4Fe-4S] cluster complex at the active site of Radical SAM enzymes (right).

Figure 2.. A mechanism for radical initiation in Radical SAM enzymes.

Inner-sphere electron transfer from the reduced [4Fe-4S] cluster to the sulfonium of SAM leads to reductive cleavage of the S-C5´ bond of SAM (left). The resulting 5´-dAdo• radical abstracts an H-atom from substrate R-H (center) to yield a substrate radical (right).

Figure 3.. Products of reductive cleavage of SAM and of anSAM.

Reductive cleavage of SAM leads to the primary carbon radical 5´-dAdo• (top), while reductive cleavage of the SAM analog anSAM leads to the allylically stabilized anAdo• radical (bottom).

Figure 4.. The organometallic intermediate Ω.

The organometallic intermediate Ω (upper left) was first discovered in PFL-AE, and the presence of the iron-carbon bond was identified by 13C-ENDOR (lower left). The Ω intermediate has since been identified for a wide range of radical SAM enzymes.

Figure 5.. Blue light-induced reductive cleavage of SAM bound to the [4Fe-4S]⁺ cluster of PFL-AE.

Blue light induces the reductive cleavage of SAM bound to the [4Fe-4S]⁺ cluster of PFL-AE, forming 5´-dAdo• cryotrapped in the active site. The 5´-dAdo• has been fully characterized through use of SAM isotopologs and EPR spectroscopy (right).

Figure 6.. Photoinduced electron transfer of SAM bound to [4Fe-4S]⁺ clusters.

Photoinduced electron transfer of SAM bound to [4Fe-4S]⁺ clusters in a range of radical SAM enzymes results in cleavage of either the S-C5´ bond to generate 5´-dAdo•, or the S-CH₃ bond to generate •CH₃, with the radicals cryotrapped in the active site (left). The regioselectivity of photoinduced S-C bond cleavage correlates with the SAM ribose ring pucker (upper right) and is explained by the Jahn-Teller effect on the one-electron reduced sulfonium radical (right).

Figure 7.. Radical intermediates observed during an adenosylation reaction catalyzed by PFL-AE on a dehydroalanine-containing substrate peptide.

The organometallic intermediate Ω is observed by rapid freeze-quench EPR at early times. Longer quench times from 10 to 18 s allows observation of both the catalytically competent 5´-dAdo• intermediate, and the adenosylated peptide radical intermediate Ado-Dha-pep•.