JAC Advance Access originally published online on April 1, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1256-1265; doi:10.1093/jac/dkn126
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Original research |
Effect of replacing glutamic residues upon the biological activity and stability of the circular enterocin AS-48
1 Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain 2 European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany
Received 22 October 2007; returned 9 February 2008; revised 18 February 2008; accepted 27 February 2008
* Corresponding author. Tel/Fax: +34-958-242857; E-mail: mmaqueda{at}ugr.es
Background: Bacteriocins are antimicrobial peptides produced by bacteria and have a relatively narrow range of activity against closely related strains. AS-48 is a circular bacteriocin produced by Enterococcus faecalis that acts against many Gram-positive and some Gram-negative bacteria, and could well serve as a natural food preservative and antimicrobial agent. The structure of AS-48 is a five-helix bundle in which a hypothetical plane containing the C
atoms of E4, E20, E49 and E58 segregates a patch of positively charged residues from the rest of the hydrophobic or uncharged surface residues.
Objectives: The aim of this study is to investigate the significance of the four glutamic residues with regard to the potency, stability and functionality of enterocin AS-48.
Methods: Four genetically engineered variants of AS-48 were obtained by replacing each glutamic residue with alanine by site-directed mutagenesis. Each mutant peptide was purified from E. faecalis cultures. The activity of highly concentrated samples and the MIC were determined against nine bacterial strains by the spot-assay method. Structural studies were made with circular dichroism (CD) spectroscopy.
Results: Occasional alterations to the net charge of AS-48 did not significantly affect its activity when high concentrations of bacteriocin were used. Nevertheless, according to the MIC values, three of the four mutated peptides showed weaker activity against the majority of the Gram-positive bacteria tested. CD spectroscopy showed that the derivatives were well structured, in a similar way to those of the native molecule, with no modifications in their helix content.
Conclusions: The spatial location of the Glu residues rather than their negative charge played a critical role in AS-48 target-cell specificity and bactericidal activity, because the replacement of Glu with Ala modify the interactions between neighbouring residues through their side chains and the interaction to the solvent affecting the protein stability and causing variations in the activity levels against identical organisms.
Keywords: bacteriocins , biological activity , circular antimicrobial peptides , site-directed mutagenesis , lactic acid bacteria , Enterococcus faecalis