Emerging themes in radical SAM chemistry

Abstract

Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe-4S] cluster coordinated to the protein via a cysteine triad motif, typically CX(3)CX(2)C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5'-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe-4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe-4S] cluster, many members of the superfamily contain additional domains and/or additional Fe-S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions.

Figure 1

The reductive cleavage of SAM catalyzed by RS enzymes. SAM is coordinated via its amino and carboxyl moieties to the unique iron of a [4Fe–4S] cluster (left). The reduced [4Fe–4S]⁺ cluster transfers an electron to SAM, thereby promoting homolytic cleavage to generate methionine and a 5′-deoxyadenosyl radical. The 5′-deoxyadenosyl radical abstracts a hydrogen atom from substrate (SH) to produce a substrate radical (S^•), methionine, and 5′-deoxyadenosine (right).

Figure 2

Crystal structures of representative RS enzymes and Dph2. The Fe–S clusters are shown in rust for iron and yellow for sulfur. SAM molecules (teal carbons) and substrates (purple carbons) are also shown. (a) PFL-AE with SAM and the substrate PFL peptide (PDB ID: 3CB8). (b) SPL with SAM and substrate dinucleoside 5R-SP (PDB ID: 4FHD). (c) BioB with SAM, DTB substrate, and the additional [2Fe–2S] cluster (PDB ID: 1R30). (d) RlmN with SAM (PDB ID: 3RFA). (e) HydE with SAM and additional [2Fe–2S] cluster (PDB ID: 3IIZ). (f) Dph2 with the iron coordinating cysteines in red (PDB ID: 3LZD).

Figure 3

Representative radical SAM reactions. (a) BioB catalyzes the sequential abstraction of hydrogen atoms from C9 and C6 of DTB (left), with insertion of sulfur from the [2Fe–2S] cluster. (b) AlbA incorporates three thioether bonds between three cysteines and the α-C of two phenylalanines and one threonine during the maturation of subtilosin A. Shown is a representative reaction between one cysteine and threonine. (c) NosL and NocL catalyze a fragmentation–recombination reaction of l-Trp to MIA which is incorporated into NOS in the case of NosL or NOC-I in the case of NocL. (d) RlmN and Cfr catalyze the methylation of A2503 of the 23S rRNA utilizing two equivalents of SAM. The sequential methylations catalyzed by these two enzymes can occur in either order.

Figure 4

Proposed biosynthesis pathway for HydA H-cluster. HydE uses an unknown substrate to synthesize a dithiolate ligand on a [2Fe–2S] cluster of HydF. HydG catalyzes the decomposition of tyrosine to produce p-cresol, CO, and CN⁻; the latter two diatomics bind to the H-cluster precursor 2Fe cluster on HydF. HydF then transfers the 2Fe H-cluster precursor to HydA to produce the complete H-cluster and the active hydrogenase. Colors of atoms are as follows: green, carbon; red, oxygen; blue, nitrogen; rust, iron; yellow, sulfur; purple, unknown bridging atom; black, unknown residue coordinating to protein.