Insights into structure-activity relationships in the C-terminal region of divercin V41, a class IIa bacteriocin with high-level antilisterial activity.
Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.
Rihakova
Jitka
J
UMR INRA SECALIM 1014 ENITIAA-ENVN, Rue de la Géraudière, BP 82225, 44322 Nantes Cedex 3, France.
Petit
Vanessa W
VW
Demnerova
Katerina
K
Prévost
Hervé
H
Rebuffat
Sylvie
S
Drider
Djamel
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001430:Bacteriocins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D013293:Enterococcus faecalis / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics; D008089:Listeria monocytogenes / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; C116372:divercin V41