Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Abstract

<jats:title>Abstract</jats:title><jats:p>Plantacyclin B21AG is a circular bacteriocin produced by <jats:italic>Lactobacillus plantarum</jats:italic> B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, <jats:italic>Listeria monocytogenes</jats:italic> and <jats:italic>Clostridium perfringens</jats:italic>. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from food grade <jats:italic>Lactobacillus</jats:italic>. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 17 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed Phe<jats:sup>8</jats:sup>Ala and Trp<jats:sup>45</jats:sup>Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. Lys<jats:sup>19</jats:sup>Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.</jats:p>

Anti-Microbial Peptides: The Importance of Structure-Function Analysis in the Design of New AMPs

“…Furthermore, the crystal structure and site directed mutagenesis of plantacyclin B21AG reveals that Phe8, Trp45 and Lys19 are essential for antimicrobial activity and a significant reduction in activity was observed with Alanine substitution mutagenesis supporting the notion of a similar role of these residues [78] (Figure 4). Moreover, many Trp rich AMPs (TrAMPs) has shown interesting antifungal activity such as synthetic peptides PW2 [79], PAF2 [80] and PEP6 [81].…”

Are specialised ABC transporters responsible for the circularisation of type I circular bacteriocins?

“…Garvicin ML is an exception however as it has been shown to bind selectively to the maltose ATP-binding cassette (ABC) transporter [16]. Phylogenetic analysis shows two distinct families of circular bacteriocins [1,3,4], and despite considerable sequence differences, they appear to demonstrate functional and structural convergence [15,[17][18][19][20], even when the precise N to C terminal ligation point is offset within the structure [1]. There are 21 circular bacteriocins reported thus far, including aureocyclicin 4185 [21], enterocin NKR-5-3B [22], amylocyclicin [23], amylocyclicin CMW1 [24], enterocin AS-48 [6], bacA [25], carnocyclin A [26], circularin A [27], thermocin 458 [28], garvicin ML [29], lactocyclicin Q [30], leucocyclicin Q [31], Pumilarin [4] uberolysin [32], acidocin B [33], butyrivibriocin AR10 [34], paracyclicin [35], gassericin A/reutericin 6 [5], plantaricyclin A [9], plantacyclin B21AG [36][37][38] and cerecyclin [3].…”