Plant defensins have attracted much attention in the development of new antimicrobials. Yet the elucidation of their modes of action against bacterial pathogens is still incipient. The available recombinant systems to obtain plant defensin mutants with enhanced or optimized antibacterial activity may help to accelerate the knowledge of their action mechanisms and their applications against pathogens. In this work, the point mutant defensin K45E (J1-1_K45E) was obtained by the same recombinant system as J1-1 defensin. The characterized peptide conserved antibacterial activity against the gram-negative Pseudomonas aeruginosa and showed a dose improvement relative to J1-1. Furthermore, the mutant J1-1_K45E exhibited a gain in function against the gram-positive Staphylococcus aureus. Finally, to correlate structural changes and antibacterial activity, two properties involved in defensins’ modes of action were measured. First, the mutant J1-1_K45E which oligomerizes in a distinct pattern was compared with J1-1 and secondly, J1-1_K45E shows a distinct lipid binding profile because it binds preferentially to phosphatidylserine. Together, our findings support the idea that amino acid sequence variability in plant defensins superfamily can generate major functional changes, and highlight the relevant role of charged residues, beyond the -core loop, in the improvement of J1-1 antibacterial activity.