Formation of an Aminovinyl-Cysteine Residue in Thioviridamide Non-Lanthipeptides Occurs through a Path Independent of Known Lanthionine Synthetase Activity in Streptomyces sp . NRRL S-87

2-minonyl-teine (AviCys) is an unusual thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs), as part of a macrocyclic ring system that contains the -terminal 4 or 6 residues of a precursor peptide. This amino acid is nonproteinogenic and arises from processing the -terminal Cys residue and an internal Ser/Thr residue to form an unsaturated thioether linkage. Enzyme activities for forming lanthionine (Lan), a distinct saturated thioether residue characteristic of lanthipeptide-related RiPPs, has long been speculated to be necessary for AviCys formation. Based on investigations into the biosynthesis of thioviridamide non-lanthipeptdes in . NRRL S-87, we here report an alternative path for AviCys formation that is independent of known Lan synthetase activity. This path relies on four dedicated enzymes for posttranslational modifications of the precursor peptide, in which TvaE, a phosphotransferase homolog, plays a critical role. It works with LanD-like flavoprotein TvaF to form a minimum AviCys synthetase complex that 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 dehydrobutyrine 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.