No abstract

Cypemycin is a parent linaridin peptide known to contain nonproteinogenic dehydrobutyrine, N,N-dimethylalanine, and aminovinyl-cysteine residues. The enzymatic process by which this ribosomally synthesized peptide is formed remains elusive largely because of the deficiency of knowledge in post-translational modifications (PTMs) conducted by CypH and CypL, the two membrane-associated enzymes unique to linaridin biosynthesis. Based on heterologous reconstitution of the pathway in Streptomyces coelicolor, we report the detailed structural characterization of cypemycin as a previously unknown, D-amino acid-rich linaridin. In particular, the unprecedented family-determining activity of CypH and CypL was revealed, which, in addition to hydrolysis for removal of the N-terminal leader peptide, leads to transformation of the core peptide part of the precursor peptide through mechanistically related 16 reactions for residue epimerization (11 amino acids), dehydration (4 Thr), and dethiolation (Cys19). Subsequent functionalization for linaridin maturation includes CypD-involved aminovinyl-cysteine formation and N,N-dimethylation of the newly exposed N-terminal D-Ala residue that requires CypM activity. Genetic, chemical, biochemical, engineering, and modeling approaches were used to access the structure of cypemycin and the versatility of the CypH and CypL combination that is achieved in catalysis. This work furthers the appreciation of PTM chemistry and facilitates efforts for expanding linaridin structural diversity using synthetic biology methods.

ABSTRACT2-Aminovinyl-cysteine (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 C-terminal 4 or 6 residues of a precursor peptide. This amino acid is nonproteinogenic and arises from processing the C-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 Streptomyces sp. 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 TvaES-87, a phosphotransferase homolog, plays a critical role. It works with LanD-like flavoprotein TvaFS-87 to form a minimum AviCys synthetase complex that follows the combined activity of TvaCDS-87 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 TvaES-87, TvaFS-87 activity for Cys processing can be coordinated with TvaCDS-87 activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.