In vivo reaction of affinity-tag-labelled epidermin precursor peptide with flavoenzyme EpiD.

The Staphylococcus epidermidis genes encoding the His-tag-labelled epidermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant protein EpiD-G93D, which lacks the coenzyme, were co-expressed and the proteins were synthesized in vivo in Escherichia coli. Only in the presence of EpiD was the precursor peptide converted to a reaction product with a decrease in mass of 44-46 Da. This result confirms the in vitro experiments carried out with purified EpiA and purified EpiD from Staphylococcus epidermidis [Kupke et al. (1994) J. Biol. Chem. 269, 5653-5659]. EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of EpiA to a [Z]-enethiol structure. In the presence of EpiD, the amount of purified (modified) peptide EpiA was several-fold higher than in the presence of EpiD-G93D, indicating that the stabilization of EpiA against proteolysis is due to an interaction with EpiD or to the presence of the C-terminal modification. The presented experimental approach will be valuable for the analysis of enzymes that catalyze posttranslational modification reaction of peptides and proteins.