Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.
Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.
Bierbaum
G
G
Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, Germany.
Szekat
C
C
Josten
M
M
Heidrich
C
C
Kempter
C
C
Jung
G
G
Sahl
H G
HG
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D001430:Bacteriocins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010455:Peptides; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D013210:Staphylococcus / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000235:genetics; D013440:Sulfides / Q000737:chemistry
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004269:DNA, Bacterial; D010455:Peptides; D013440:Sulfides; C054222:lantibiotic Pep5