EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF EPIC, AN ENZYME INVOLVED IN THE BIOSYNTHESIS OF THE LANTIBIOTIC EPIDERMIN, AND SEQUENCE-ANALYSIS OF STAPHYLOCOCCUS-EPIDERMIDIS EPIC MUTANTS

The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tu3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identifi ed by Western blotting (immunoblotting) with an anti-EpiC-peptide anti serum. EpiC was a hydrophobic but soluble protein. EpiC was purified b y hydrophobic-interaction chromatography. The determined amino-termina l amino acid sequence was M I N I N N I.... The electrophoretic migrat ion behavior of EpiC depended on the oxidation state of the enzyme, in dicating the formation of an intramolecular disulfide bridge between C -274 and C-321. The cysteine residues in the motifs WC-274YG and C-321 HG of EpiC are conserved in all lantibiotic enzymes of the C type (so- called LanC proteins) and in the CylM protein. Mutated epiC genes from S. epidermidis epiC mutants were cloned and expressed in E. coli. Seq uence analysis revealed that the mutations occurred in the two motifs -S-X-X-X-G-X-X-G- and -N-X-G-X-A-H-G-X-X-G-, which are conserved in al l LanC proteins. For the investigation of EpiC-EpiA interactions, prec ursor peptide EpiA was coupled to N-hydroxysuccinimide-activated Sepha rose High Performance material (HiTrap). Under reducing conditions, Ep iC was retarded on the EpiA-HiTrap column. In the incubation experimen ts, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides.