The cypemycin decarboxylase CypD is investigated by using a synthetic oligopeptide, which contains the to-be-cyclized dehydroalanine (Dha) residue. It was shown that CypD efficiently catalyzes the decarboxylation of this Dha-containing peptide, but the expected AviCys ring is not formed in the product, suggesting that CypD alone is not enough to form the AviCys ring. It was also shown that the Dha-containing peptide is a better substrate than two similar peptides with a Ser or a Cys residue, supporting that, in cypemycin biosynthesis, Dha formation is prior to decarboxylation of the C-terminal Cys.