P. Chen et al., STRUCTURE-ACTIVITY STUDY OF THE LANTIBIOTIC MUTACIN-II FROM STREPTOCOCCUS-MUTANS-T8 BY A GENE REPLACEMENT STRATEGY, Applied and environmental microbiology, 64(7), 1998, pp. 2335-2340
Mutacin II, elaborated by group II Streptococcus mutans, is a ribosoma
lly synthesized and posttranslationally modified polypeptide antibioti
c containing unusual thioether and didehydro amino acids, To ascertain
the role of specific amino acid residues in mutacin II antimicrobial
activity, we developed a streptococcal expression system that facilita
tes the replacement of the mutA gene with a single copy of a mutated v
ariant gene. As a result, variants of mutacin II can be designed and e
xpressed, The system was tested by constructing the following mutant p
eptides: Delta N1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A, All of
these mutacin II variants except Delta N1 and T10A, which were not sec
reted, were isolated, and their identities were verified by mass spect
rometry, Variants P9A, C15A, C26A, and C27A failed to exert antimicrob
ial activity, Because the P9A and T10A variants comprise the ''hinge''
region of mutacin II, these observations suggest that in addition to
the thioether and didehydro amino acids, the hinge region is essential
for biological activity and biosynthesis or export of the peptide. Ta
ndem mass spectrometry of the N-terminal part of the wild-type molecul
e and its C15A variant confirmed that the threonine at position 10 is
dehydrated and present as a didehydrobutyrine residue. This analysis o
f the active T10S variant further suggested that a didehydro amino aci
d at this position is specific for antimicrobial activity and that the
biosynthetic machinery does not discriminate between threonine and se
rine, In contrast, the lack of production of mutacin variants with ala
nine substituted for threonine at position 10, as well as the deletion
of asparagine at the N terminus (Delta N1), indicates that specific r
esidues in the propeptide may be crucial for certain steps in the bios
ynthetic pathway of this lantibiotic.