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

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.

Dissection and Modulation of the Four Distinct Activities of Nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation

“…Moreover, gallidermin is approximately 10-fold more effective in cell killing than nisin, which might be related to a more-efficient inhibition of cell wall biosynthesis. Ring C, the hinge region, and the C terminus of nisin have been subjected to a limited mutational analysis (8,22,39). Interestingly, the N20K and M21K mutants displayed antimicrobial activities against gram-negative Shigella, Pseudomonas, and Salmonella species (43).…”

“…The opening of ring A strongly reduces antimicrobial activity against Micrococcus luteus NCDO 8166 and leads to a complete loss of antimicrobial activity against Lactococcus lactis MG1614 (8). Only a few conservative ring A mutants (S3T, which has reduced activity [21]; S5A [7]; S5T [22]; and S5C [39]) and chemically modified variants (33) have been described, whereas no reports on ring B mutants have appeared. Here, we randomized the amino acids of these functionally important rings to investigate the possibility of engineering nisin mutants with improved or altered characteristics.…”

Isolation and Identification of a Paenibacillus polymyxa Strain That Coproduces a Novel Lantibiotic and Polymyxin

“…These modifications generate dehydrated amino acids, i.e., ␣,␤-didehydroalanine (Dha) and ␣, ␤-didehydrobutyric acid (Dhb) and thioether bridges of lanthionine (Lan) and ␤-methyllanthionine (MeLan), as well as some other less frequently encountered modifications (6). These modified residues are believed to stabilize molecular conformations that are essential for the antimicrobial activity of lantibiotics and their resistance to proteases of the producing strains (3,29,41). It is noteworthy that the lantibiotics produced by lactic acid bacteria have been tested as biopreservatives in a number of food products (10)(11)(12)26), with nisin being the most prominent member of these bacteriocins.…”

Complete Covalent Structure of Nisin Q, New Natural Nisin Variant, Containing Post-Translationally Modified Amino Acids

“…4), since some previous studies indicate that the locations of the unusual amino acids in some lantibiotics play crucial roles in antimicrobial properties. [19][20][21][22][23] Recently, a fourth nisin variant, nisin U, was discovered, 24) suggesting that further screening might reveal more nisin variants. This is the first study to elucidate the complete covalent structure of nisin without decomposition.…”