Biochemical structural analysis of the lantibiotic mutacin II.
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 NRWWQGVVPTVSYECRMNSWQHVFTCC, 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.
Krull
R E
RE
Department of Oral Biology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Chen
P
P
Novak
J
J
Kirk
M
M
Barnes
S
S
Baker
J
J
Krishna
N R
NR
Caufield
P W
PW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001430:Bacteriocins / Q000737:chemistry*; D003488:Cyanogen Bromide; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D015202:Protein Engineering; D020539:Sequence Analysis, Protein; D013295:Streptococcus mutans / Q000737:chemistry* / Q000235:genetics; D013440:Sulfides / Q000737:chemistry
D001426:Bacterial Proteins; D001430:Bacteriocins; D010446:Peptide Fragments; D013440:Sulfides; C082508:lacticin 481; C069066:mutacin II; D003488:Cyanogen Bromide