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 Tü3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration behavior of EpiC depended on the oxidation state of the enzyme, indicating the formation of an intramolecular disulfide bridge between C-274 and C-321. The cysteine residues in the motifs WC-274YG and C-321HG 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. Sequence 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 all LanC proteins. For the investigation of EpiC-EpiA interactions, precursor peptide EpiA was coupled to N-hydroxysuccinimide-activated Sepharose High Performance Material (HiTrap). Under reducing conditions, EpiC was retarded on the EpiA-HiTrap column. In the incubation experiments, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides.
Kupke
T
T
Mikrobielle Genetik, Universität Tübingen, Federal Republic of Germany.
Gotz
F
F
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001430:Bacteriocins; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013212:Staphylococcus epidermidis / Q000201:enzymology / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; C098712:EpiA protein, Staphylococcus epidermidis; C098713:EpiC protein, Staphylococcus epidermidis; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C050558:epidermin