Peptide AS-48: prototype of a new class of cyclic bacteriocins

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.