Engineering a disulfide bond and free thiols in the lantibiotic nisin Z

The antimicrobial peptide nisin contains the uncommon amino acid residues l anthionine and methyl-lanthionine, which are post-translationally formed fr om Ser, Thr and Cys residues. To investigate the importance of these uncomm on residues for nisin activity, a mutant was designed in which Thr13 was re placed by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfi de bridge, a bond that is very unusual in lantibiotics. NMR analysis of thi s 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 dra matic reduction in antimicrobial activity to less than 1% of wild-type acti vity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and character ized; 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 t hiol. Secretion of these peptides by the lactococcal producer cells, as wel l 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 prop erties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.