Biochemical Structural Analysis of the Lantibiotic Mutacin II

Abstract

Mutacin II is a post-translationally modified lantibiotic peptide secreted by Streptococcus mutans T8, which inhibits the energy metabolism of sensitive cells. The deduced amino acid sequence of promutacin II is NRW-WQGVVPTVSYECRMNSWQHVFTCC, which is capable of forming three thioether bridges. It was not obvious, however, how the three thioether bridges are organized. To examine the bridging, the cyanogen bromide cleavage products of mutacin II and its variants generated by protein engineering, C15A, C26A, and C15A/C26A, were analyzed by mass spectrometry. Analysis of the wild type molecule and the C15A variant excluded several possibilities and also indicated a high fidelity of formation of the thioether bridges. This allowed us to further resolve the structure by analysis (mass spectrometry and tandem mass spectrometry) of the cyanogen bromide cleavage fragments of the C26A and C15A/C26A mutants. Nuclear magnetic resonance analysis established the presence of one and two dehydrobutyrine residues in mutacin II and the C15A variant, respectively, thus yielding the final structure. The results of this investigation showed that the C-terminal part contains three thioether bridges connecting Cys residues 15, 26, and 27 to Ser/Thr residues 10, 12 and 19, respectively, with Thr 25 being modified to dehydrobutyrine.

Ruminococcin A, a New Lantibiotic Produced by a Ruminococcus gnavu s Strain Isolated from Human Feces

“…It had been shown previously that MS/MS detection signals corresponding to Dhb may vary, depending on whether the residues are involved in Me-Lan structures or remain unreacted (16). In our experiments, MS/MS signals corresponding to both Dhb 7 and Dhb 16 were comparable.…”

“…In our experiments, MS/MS signals corresponding to both Dhb 7 and Dhb 16 were comparable. As the presence of Me-Lan structures generally leads to blockages during recurrent degradation of lantibiotics (26), these observations strongly suggested that both didehydro residues remained unreacted in RumA.…”

“…Again, these data were consistent with the RumA Cterminal sequence obtained by the Edman degradation method (Table 3). They made it possible to identify the 16th residue as a Dhb (Dhb 16 ). It was noticeable that the signals corresponding to Dhb 7 and DHb 16 were comparable, suggesting that the two residues were at the same stage.…”

“…They made it possible to identify the 16th residue as a Dhb (Dhb 16 ). It was noticeable that the signals corresponding to Dhb 7 and DHb 16 were comparable, suggesting that the two residues were at the same stage. From these results, it could also be deduced that this last fragment was an internal sequence of RumA, since the molecular mass difference between m/z 1338 and 1264, i.e., 148 Da, corresponded to the M r of Phe minus 17 Da (OH group), underlining the fact that this amino acid, i.e., Phe 21 , was naturally involved in two peptide bonds and hence demonstrating that it was not the last C-terminal residue of RumA.…”

Bacteriocin Diversity in Streptococcus and Enterococcus

“…Mutacin II has been shown to be structurally related to the lacticin 481 group of type A lantibiotics (73), while the structures of mutacins I and III have not been determined. A two-peptide class II peptide bacteriocin (mutacin IV) is produced by S. mutans UA140, a strain that also produces mutacin I (100).…”