No abstract

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

SummaryLantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to sitedirected mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a twocomponent lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibioticbased antimicrobial compounds.

As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA͞RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and͞or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).antimicrobial ͉ bacteriocin ͉ chirality T he presence of D-amino acids in ribosomally synthesized peptides was first observed in dermorphins, peptides found in the skin secretions of species of a subfamily of South American tree frogs, Phyllomedusinae. Subsequently, D-amino acids have been identified in a number of other ribosomally synthesized peptides of eukaryotic origin (1, 2). -Agatoxin IVb and bombinin H, produced by the funnel-web spider and the skin secretions of the frog Bombina variegata, respectively, represent the only instances in which the responsible enzymes, both peptide epimerases, have been identified (1, 2). For -agatoxin IVb the relevant L-serine to D-serine conversion is thought to involve a deprotonation͞reprotonation reaction (3). The presence of D-amino acids in many of these peptides is crucial, as demonstrated by the creation of synthetic analogues with Lrather than D-amino acids at the relevant locations, which are devoid of activity (4). The artificial incorporation of D-amino acids can also be beneficial. For example, a growing number of synthetic peptides͞peptide libraries containing D-amino acids have been assembled to capitalize on the ability of such residues to provide improved protease stability [enhanced pharmokinetic profile (5-7)], alter tertiary structure (new structural elements can be assembled that cannot be built solely from L-amino acids), and affect the activity of bioactive peptides [enhanced antibacterial activity with concomitant reduction in cell cytotoxicity (5, 6, 8-11)].In prokaryotic systems, there are only a few examples of L-to D-amino acid conversions. It...