Studies of nisin dehydro residue reactivities and mutagenesis of sublancin dehydro residues

Nisin and sublancin are small peptides belonging to the lantibiotic family of antimicrobial peptides. Lantibiotics are gene-encoded and ribosomally synthesized as prepeptides, then posttranslationally modified, proteolytically activated and exported out of the cell. The posttranslational modifications dehydrate serines and threonines, leading to the creation of four unusual amino acids: dehydroalanine, dehydrobutyrine, lanthionine and methyllanthionine. These amino acids give lantibiotics their unusual properties.; The three dehydrated, non-ring forming residues of nisin bind cysteine in a pH dependent manner. The binding was demonstrated by mass spectrometry which showed that the mass of nisin increased by up to three cysteines. The binding was also demonstrated by NMR spectrometry which showed that the three peaks representing the three dehydrated residues disappeared or were significantly suppressed by the addition of cysteine. The rates of disappearance and appearance of nisin and the nisin-cysteine adducts were measured by mass spectrometric quantitation of the four nisin-cysteine adducts at time points during the reaction. The experiment was done at pH 7.0, 8.3 and 9.3. The actual rates of reaction for each dehydro residue were determined by fitting this data to a proposed reaction scheme. These rates were assigned to the appropriate residue by NMR spectrometry of the reaction mixture which demonstrated which residues disappeared at what rate relative to others. The forward and reverse rates at pH 8.3 were 0.56 and 0.01 for Dha5, 0.42 and 0.07 for Dha33 and 0.06 and 0.12 for Dhb2.; Three sublancin mutants were constructed using the pLPcat vector system: S16A sublancin, S16T sublancin and NRM sublancin, in which residues 19–21 of S16A sublancin were replaced with Ser-Ile-Ser-Leu. The S16A and S16T mutants were correctly processed and exported. The mutations were confirmed by mass spectrometric peptide sequencing. S16A and S16T sublancin were active against Bacillus cereus T spores. S16A sublancin lost activity against Staphylococcus aureus and exhibited enhanced activity relative to sublancin against Bacillus subtilis 6633 and Bacillus megaterium. S16T sublancin lost activity against S. aureus and exhibited activity comparable to sublancin against other strains tested. NRM sublancin was not correctly processed and was poorly exported. Genomic DNA sequencing confirmed sunA was correctly modified to produce the desired prepeptide. NRM sublancin was not active against B. cereus T spores or S. aureus and exhibited decreased activity against B. subtilis 6633. NRM sublancin exhibited novel activity against B. megaterium. These results suggest sublancin has three modes of action. One depends at least on Dha16 for activity against S. aureus. Another depends on ring C and/or sublancin's 197 Da adduct for activity against B. cereus T spores and vegetative cells. The third action, activity against other vegetative cells, does not require Dha16 or ring C and/or the 197 Da adduct. Thus, the ability to incorporate new activities into lantibiotics was demonstrated by NRM sublancin.