1. Engineering a disulfide bond and free thiols in the lantibiotic nisin Z
- Author
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van Kraaij, C, Breukink, E, Rollema, HS, Bongers, RS, Kosters, HA, de Kruijff, B, Kuipers, OP, Rollema, Harry S., Bongers, Roger S., Groningen Biomolecular Sciences and Biotechnology, and Molecular Genetics
- Subjects
Lactococcus lactis ,IDENTIFICATION ,CYSTEINE-SCANNING MUTAGENESIS ,RESIDUES ,nisin ,protein engineering ,BIOSYNTHESIS ,C-TERMINAL PART ,MEMBRANE ,LACTOCOCCUS-LACTIS ,cysteine ,CHEMICAL MODIFICATION ,mutagenesis - Abstract
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.
- Published
- 2000