Engineering a disulfide bond and free thiols in the lantibiotic nisin Z
2000
van Kraaij, Cindy | Breukink, Eefjan | Rollema, Harry S. | Bongers, Roger S. | Kosters, Hans A. | de Kruijff, Ben | Kuipers, O. P.
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.
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