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.
Kupke
T
T
Universität Tübingen, Germany.
Götz
F
F
D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins; D001430:Bacteriocins; D001483:Base Sequence; D002262:Carboxy-Lyases; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010455:Peptides; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; C098712:EpiA protein, Staphylococcus epidermidis; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D006639:Histidine; C050558:epidermin; D010088:Oxidoreductases; C075971:epidermin decarboxylase; D002262:Carboxy-Lyases