flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide (SFNSYCC7)-F-1 of EpiA were used for determining the substrate specificity of EpiD, When Cys(7) was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed, Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate (SFNSYCC7)-F-1 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry, When the penultimate cysteine residue Cys(6) of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred, Tyr(5) could be replaced with other hydrophobic amino acid residues, Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides, Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified, The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products, To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.