Covalent structure, synthesis, and structure-function studies of mesentericin Y 10537, a defensive peptide from gram-positive bacteria leuconostoc mesenteroides

Abstract

A 37-residue cationic antimicrobial peptide named mesentericin Y 10537 was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVN-WGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 10537 contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 10537 was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 10537 is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 10537 in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% α-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 10537 targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum α-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 10537 are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative α-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 10537 is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.