IN-VIVO REACTION OF AFFINITY-TAG-LABELED EPIDERMIN PRECURSOR PEPTIDE WITH FLAVOENZYME EPID

The Staphylococcus epidermidis genes encoding the His-tag-labelled epi dermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant p rotein EpiD-G93D, which lacks the coenzyme, were co-expressed and the proteins were synthesized in vivo in Escherichia coli. Only in the pre sence of EpiD was the precursor peptide converted to a reaction produc t with a decrease in mass of 44-46 Da. This result confirms the in vit ro experiments carried out with purified EpiA and purified EpiD from S taphylococcus epidermidis [Kupke et al. (1994) J. Biol. Chem. 269, 565 3-5659]. EpiD catalyzes the oxidative decarboxylation of the C-termina l cysteine residue of EpiA to a [Z]-enethiol structure. In the presenc e of EpiD, the amount of purified (modified) peptide EpiA was several- fold higher than in the presence of EpiD-G93D, indicating that the sta bilization of EpiA against proteolysis is due to an interaction with E piD or to the presence of the C-terminal modification. The presented e xperimental approach will be valuable for the analysis of enzymes that catalyze posttranslational modification reactions of peptides and pro teins.