Biosynthesis of Aminovinyl-Cysteine-Containing Peptides and Its Application in the Production of Potential Drug Candidates

Bacteria produce a wide array of metabolites to protect them selves from competing microbes. These antimicrobial compounds include peptides with an S-[(Z-2-aminovinyl]-D-cysteine (AviCys) or S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine (AviMeCys) residue, which have been isolated from several different bacterial species. The peptides are structurally diverse: some feature polycyclic backbones, such as the lantibiotic epidermin, and others feature a mostly linear structure, such as cypemycin. Each of the AviCys-containing peptides characterized to date exhibit highly potent biological activities, ranging from antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) to anticancer activity against mouse leukemia cells. The AviCys-containing peptides gallidermin and mutacin 1140 have been suggested as possible treatments of acne and of throat infections, respectively. Unfortunately, their low production yield in fermentation (typically only 10-200 mg/L) remains a major hindrance to the widespread use and clinical testing of AviCys-containing peptides for human therapeutics. Although scientists have made great strides in the total chemical synthesis of polycyclic peptides on solid support, an efficient method to form the AviCys ring has yet to be developed. In light of these difficulties, it may be possible to draw inspiration from the natural biosynthesis of AviCys-containing peptides within the producer organisms. In this Account, we examine the characteristics of the enzymes responsible for constructing AviCys to evaluate possibilities for generating high yields of bioactive AviCys- or AviMeCys-containing peptides for research and clinical use. The gene cluster for the biosynthesis of epidermin has been studied in depth, leading to the proposal for a mechanism of AviCys formation. First, a serine residue upstream of the C-terminus is enzymatically dehydrated to form a dehydroalanine residue. Then, the C-terminal cysteine residue is oxidatively decarboxylated to form an enethiolate, which subsequently cyclizes onto the dehydroalanine to give the AviCys ring. Extensive research on EpiD, the enzyme responsible for the oxidative decarboxylation reaction, has led to its purification and cocrystallization with a model substrate peptide, yielding an X-ray crystal structure. An in vitro assay of the enzyme with a library of synthetic heptapeptides has resulted in the discovery that EpiD has low absolute substrate specificity and can oxidatively decarboxylate a wide variety of C-terminal cysteine-containing peptides. Recently, the gene duster for the biosynthesis of cypemycin was also identified. Despite certain structural similarities between cypemycin and the lantibiotic peptides, analysis of the biosynthetic genes suggests that cypemycin production is quite different from that of the lantibiotics. In particular, the AviCys residue in cypemycin is formed from two cysteine residues instead of one serine and one cysteine, and the CypD enzyme that catalyzes the oxidative decarboxylation of the C-terminal cysteine shows little homology to EpiD. The knowledge accrued from studying EpiD and CypD could be used to develop a semisynthetic methodology to produce AviCys-containing peptides. In particular, suitable precursor peptides could be synthesized on solid support before being fed to either of these enzymes in vitro to generate the C-terminal AviCys moiety. Exploring the potential of this methodology could lead to the efficient production of epidermin, cypemycin, and analogues thereof.