Search

The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ~700K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymatic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymology strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymatic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their experimental studies to discover new enzymatic activities and metabolic functions, (ii) contribute experimentally verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the β- or γ-carbon (i.e. non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X(3)-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the β-carbon of Asp (S-Cβ). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant towards Ala and Asn in the acceptor position but will tolerate Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-X(n)-Asp, n=2,4), although a minimal substrate featuring only one Cys-X(3)-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which lead to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.