Structural Insights into Radical Generation by the Radical SAM Superfamily

Abstract

Chemical Reviews REVIEW(DFT) 36 studies recently focusing attention on iron as the likely mediator of electron transfer.Recently, the radical SAM superfamily has been further expanded by the characterization of ThiC. [37][38][39][40] While studying thiamine pyrimidine biosynthesis, Downs et al. found that the protein ThiC carries out radical SAM chemistry but does not contain the conserved CX 3 CXφC motif. 37,41 Although structures of the apo protein (with disordered cluster binding loop) are available, 39 comparison with other radical SAM enzyme structures awaits crystallographic analysis of an iron-sulfur cluster-bound form. Further, similar 4Fe-4S-dependent radicalization of SAM has been discovered in a new enzymatic fold with the characterization of Dph2 of the diphthamide biosynthetic pathway. [42][43][44] Although we will only discuss ThiC and Dph2 briefly in this review, these findings suggest that the predicted >2800 unique sequences 6 already assigned to the CX 3 CXφC-containing radical SAM superfamily may need to make room for an unknown number of additional radical SAM enzymes.

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…Two [4Fe-4S] clusters are located within the TIM barrel and are bound by cysteine triads [14]. Comparison with other RS enzymes [40] indicates the cluster responsible for the reductive cleavage of SAM, [4Fe-4S] RS , is bound to cysteines 63, 67 and 70 on the loop between strand β-1 and helix α-1. The auxiliary cluster, [4Fe-4S] Aux is bound by three residues from the N-terminal extension, cysteines 37, 42 and 48.…”

“…DTT is bound to directly to the unique iron of the [4Fe-4S] RS through one of the thiols (Figure 2A) and therefore occupies the ligand site typically occupied by the methionine moiety of SAM. As a result, the 'GGE' sequence motif, which in other RS enzymes forms hydrogen bonds to the SAM amino group [19,40], has no H-bonding partner in the TeLipA2:MTA structure. Two motifs shared by RS enzymes and previously described as recognizing SAM [40,44] form interactions with the MTA (Figure 3C).…”

“…As a result, the 'GGE' sequence motif, which in other RS enzymes forms hydrogen bonds to the SAM amino group [19,40], has no H-bonding partner in the TeLipA2:MTA structure. Two motifs shared by RS enzymes and previously described as recognizing SAM [40,44] form interactions with the MTA (Figure 3C). Firstly, the 'ribose motif' includes residues Asn169 and Glu171 which are positioned to form hydrogen bonds to the 2' and 3' hydroxyl groups of the ribose ring.…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…The [4Fe-4S] RS clusters of monomers A and B are coordinated by the α-amino and α-carboxy groups of methionine and SAM respectively. The SAM sulfonium sulfur atom is poised 3.4 Å from the proximal iron, an arrangement that closely resembles SAM binding in other radical SAM enzymes [23]. The structure of co-crystals of HydG with tyrosine have not yet been reported, but this complex has been modelled [20] with the tyrosine binding pocket adjacent to the 5ʹ-C of SAM (Figure 2b).…”

“…The [4Fe-4S] RS clusters are embedded within canonical sequence motifs shared by members of the radical SAM family [23]. The [4Fe-4S] RS clusters of monomers A and B are coordinated by the α-amino and α-carboxy groups of methionine and SAM respectively.…”