Effect of amino acid substitutions on the activity of carnobacteriocin B2. Overproduction of the antimicrobial peptide, its engineered variants, and its precursor in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 272; no. 6; pp. 3384 - 3388
• Main Authors: Quadri, L.E.N. (LHRRB, Boston, MA.), Yan, L.Z, Stiles, M.E, Vederas, J.C
• Format: Journal Article
• Language: English
• Published: United States 07-02-1997
• Subjects: Amino Acid Sequence; AMINO ACID SEQUENCES; Amino Acids - chemistry; ANTIBACTERIAL PROPERTIES; ANTIMICROBIAL PROPERTIES; ATP-Binding Cassette Transporters; Bacterial Proteins - biosynthesis; BACTERIAS ACIDOLACTICAS; BACTERIE LACTIQUE; BACTERIOCINAS; BACTERIOCINE; BACTERIOCINS; Bacteriocins - biosynthesis; BINDING PROTEINS; CARNOBACTERIUM PISCICOLA; Carrier Proteins - biosynthesis; CHEMICAL COMPOSITION; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; ESCHERICHIA COLI; Escherichia coli Proteins; EXPRESION GENICA; EXPRESSION DES GENES; GENE EXPRESSION; INDUCED MUTATION; LACTIC ACID BACTERIA; MALTOSE BINDING PROTEIN; Maltose-Binding Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; MUTACION INDUCIDA; MUTANT; MUTANTES; MUTANTS; MUTATION PROVOQUEE; PROPIEDADES ANTIMICROBIANAS; PROPRIETE ANTIMICROBIENNE; Protein Precursors - biosynthesis; PROTEIN SYNTHESIS; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; PROTEINS; Recombinant Fusion Proteins - biosynthesis; RECOMBINANT PROTEINS; Sequence Alignment; SINTESIS DE PROTEINAS; Structure-Activity Relationship; SYNTHESE PROTEIQUE
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.6.3384

Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola. Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr3 leads replaced by Phe), CbnS33 (Phe33 replaced by Ser), CbnI34 (Val34 replaced by Ile), CbnI37 (Val37 replaced by Ile), CbnG46 (Arg46 replaced by Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) + ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac- cultures and reestablish the Bac+ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus