Formation of an aminovinyl-cysteine residue in thioviridamides occurs through a path independent of known lanthionine synthetase activity.

2-Aminovinyl-cysteine (AviCys) is a thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on investigations into the biosynthesis of thioviridamide RiPPs in Streptomyces sp. NRRL S-87, we here report a path for the formation of this unusual thioether residue. This path relies on four dedicated proteins: phosphotransferase TvaC, Lyase TvaD, kinase homolog TvaE, and LanD-like flavoprotein TvaF. TvaE plays a critical role in effective AviCys formation. During the posttranslational modifications of the precursor peptide, it works with TvaF to form a minimum AviCys synthetase complex, which follows the combined activity of TvaCD for Thr dehydration and catalyzes Cys oxidative decarboxylation and subsequent Michael addition of the resulting enethiol nucleophile onto the newly formed dehydroamino acid residue for cyclization. With TvaE, TvaF activity for Cys processing can be coordinated with TvaCD activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.