Reconstitution and Substrate Specificity of the Thioether-Forming Radical -Adenosylmethionine Enzyme in Freyrasin Biosynthesis.

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 -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 , which contains six thioethers from Cys-X-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 toward Ala and Asn in the acceptor position but tolerates 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-Asp, = 2 or 4), although a minimal substrate featuring only one Cys-X-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 . The activity of PapB could be restored by supplying the excised RRE . Finally, we reconstituted the activity of PapB , which led 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.