Aromaticity of Tyr-202 in the alpha 4-alpha 5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin
Autor(es): Pornwiroon, W.; Katzenmeier, G.; Panyim, S.; Angsuthanasombat, C.
Resumo: The current model for the mechanism of action of the Bacillus thuringiensis Cry delta-endotoxins involves the penetration of the alpha4-alpha5 hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the alpha4-alpha5 loop of the 130-kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the alpha4-alpha5 loop were performed. Like the wildtype, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the alpha4-alpha5 loop of the Cry4A toxin.
Palavras-Chave: Aromaticity; Bacillus thuringiensis; Delta-endotoxin; Alpha 4-alpha 5 loop; Larvicidal activity; Mutagenesis
Imprenta: Journal of biochemistry and molecular biology , v. 37, n. 3, p. 292-297, 2004
Identificador do objeto digital: 10.5483/BMBRep.2004.37.3.292
Descritores: Aedes aegypti - Cell ; Aedes aegypti - Proteins
Data de publicação: 2004
