The Radical S-Adenosyl-l-methionine Enzyme MftC Catalyzes an Oxidative Decarboxylation of the C-Terminus of the MftA Peptide

Abstract

Ribosomally synthesized post-translationally modified peptides (RiPPs) are encoded in the genomes of a wide variety of microorganisms, in the proximity of open reading frames that encode enzymes that conduct extensive modifications, many of which are novel. Recently, members of the radical S-adenosyl-l-methionine (SAM) superfamily have been identified in these biosynthetic clusters. Herein, we demonstrate the putative radical SAM enzyme, MftC, oxidatively decarboxylates the C-terminus of the MftA peptide in the presence of the accessory protein MftB. The reaction catalyzed by MftC expands the repertoire of peptide-based radical SAM chemistry beyond the intramolecular cross-links.

Figure 1

(A) Extracted ion chromatogram corresponding to dAdo (m/z = 251.5–252.5). dAdo is only formed when MftC and SAM are present in the reaction. MftB has no effect on the formation of dAdo showing that MftC alone is capable of catalyzing the reductive cleavage of SAM. (B) Mass spectrum of dAdo eluting ca. 15 min.

Figure 2

The mass spectrum zoomed in on the +2 charge state mass envelope and the corresponding deconvoluted mass spectrum of unlabeled MftA isolated from reactions where either SAM (A and B), MftB (C and D), or MftC (E and F) were omitted. G and H correspond to the peptide isolated from the reaction where unlabeled MftA was incubated in the presence of all three components. I and J correspond to the peptide isolated from the reaction where [¹³C₉,¹⁵N]-Tyr₃₀ MftA was incubated in the presence of MftB, MftC, and SAM. The blue and red boxes highlight the peaks corresponding to unmodified and modified MftA respectively. The deconvoluted mass spectra were generated from the full mass spectra shown in Fig S6.

Figure 3

Proposed catalytic mechanism for the oxidative decarboxylation of the C-terminus of MftA catalyzed by MftC.

Scheme 1

Mycofactocin gene cluster from Mycobacterium smegmatis ATCC 700084. The sequence of the peptide encoded by the mftA gene is shown.