Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin.
The plasmid-encoded epidermin biosynthesis gene, epiD, of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using both the malE fusion system and the T7 RNA polymerase-promoter system. EpiD was identified by Western blotting (immunoblotting) with anti-maltose-binding protein (MBP)-EpiD antiserum. EpiD and the MBP-EpiD fusion protein, which were mainly present in the soluble protein fraction, were purified from the respective E. coli clones. Purified EpiD showed the typical absorption spectrum of an oxidized flavoprotein with maxima at 274, 382, and 453 nm. The coenzyme released from EpiD by heat treatment was identified as flavin mononucleotide. S. epidermidis Tü3298/EMS11, containing a mutation within epiD, was unable to synthesize active epidermin. This mutated gene, epiD*, was cloned in E. coli and expressed as an MBP-EpiD* fusion protein. DNA sequencing of epiD* identified a point mutation that led to replacement of Gly-93 with Asp. Unlike MBP-EpiD, the fusion protein MBP-EpiD* could not bind flavin mononucleotide. We propose that EpiD catalyzes the removal of two reducing equivalents from the cysteine residue of the C-terminal meso-lanthionine to form a --C==C-- double bond and is therefore involved in formation of the unusual S-[(Z)-2-aminovinyl[-D-cysteine structure in epidermin.
Kupke
T
T
Mikrobielle Genetik, Universität Tübingen, Germany.
Stevanović
S
S
Sahl
H G
HG
Götz
F
F
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001430:Bacteriocins; D001483:Base Sequence; D015153:Blotting, Western; D002262:Carboxy-Lyases; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D010455:Peptides; D010456:Peptides, Cyclic / Q000096:biosynthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013212:Staphylococcus epidermidis / Q000235:genetics*; D014764:Viral Proteins
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D004269:DNA, Bacterial; D005420:Flavoproteins; D010455:Peptides; D010456:Peptides, Cyclic; D014764:Viral Proteins; C050558:epidermin; D010088:Oxidoreductases; C075971:epidermin decarboxylase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D002262:Carboxy-Lyases