Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.
The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.
van Kraaij
C
C
NIZO Food Research, Section Flavours and Natural Ingredients, Ede, The Netherlands. vankraay@nizo.nl
Breukink
E
E
Rollema
H S
HS
Bongers
R S
RS
Kosters
H A
HA
de Kruijff
B
B
Kuipers
O P
OP
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D013294:Lactococcus lactis / Q000187:drug effects / Q000235:genetics; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009561:Nisin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010539:Permeability; D015202:Protein Engineering; D013291:Streptococcus / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004220:Disulfides; D008081:Liposomes; D013438:Sulfhydryl Compounds; D009561:Nisin; D003545:Cysteine; C071364:nisin Z