Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity

• Published in: The Journal of biological chemistry Vol. 272; no. 2; pp. 1171 - 1179
• Main Authors: Samblanx, G.W, Goderis, I.J, Thevissen, K, Raemaekers, R, Fant, F, Borremans, F, Acland, D.P, Osborn, R.W, Patel, S, Broekaert, W.F
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.01.1997
• Subjects: AGENT PATHOGENE; Amino Acid Sequence; AMINO ACID SEQUENCES; Antifungal Agents - chemistry; ANTIFUNGAL PROPERTIES; BIOLOGICAL DIFFERENCES; Blood Proteins - genetics; CALCIO; CALCIUM; CATION; CATIONES; CATIONS; CHEMICAL COMPOSITION; Circular Dichroism; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DEFENCE MECHANISMS; Defensins; DIFERENCIAS BIOLOGICAS; DIFFERENCE BIOLOGIQUE; DNA, Plant - chemistry; ENFERMEDADES FUNGOSAS; FUNGAL DISEASES; FUSARIUM CULMORUM; GRAINE; INDUCED MUTATION; ION; ION UPTAKE; IONES; IONS; MALADIE FONGIQUE; MECANISME DE DEFENSE; MECANISMOS DE DEFENSA; MICELIO; Models, Molecular; MOLECULAR SEQUENCE DATA; MUTACION; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTANTS; MUTATION; MUTATION PROVOQUEE; MYCELIUM; ORGANISMOS PATOGENOS; PATHOGENS; PLANT PROTEINS; POTASIO; POTASSIUM; PROPIEDADES ANTIMICOSICAS; PROPRIETE ANTIFONGIQUE; Protein Conformation; PROTEINAS; PROTEINAS RECOMBINANTES; PROTEINE; PROTEINE RECOMBINANTE; PROTEINS; RAPHANUS SATIVUS; RECOMBINANT PROTEINS; SEEDS; SEMILLA; SPECIES DIFFERENCES; TARGETED MUTAGENESIS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.2.1171

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI β-turn connecting β-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting β-strand 1 and the α-helix and contiguous residues on the α-helix and β-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca 2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca 2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca 2+ uptake.