Specific mutations within the 4-5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170
Autor(es): Kanintronkul, Yodsoi; Sramala, Issara; Katzenmeier, Gerd; Panyim, Sakol; Angsuthanasombat, Chanan
Resumo: The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry d-endotoxins suggests that helices 4 and 5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the 4-5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-b-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within thea 4-5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.
Palavras-Chave: Aromaticity; Bacillus thuringiensis; ?-endotoxins; Mutagenesis; Larvicidal activity; Polarity
Imprenta: Molecular Biotechnology, V. 24, n. 1, p. 11-19, 2003.
Descritores: Aedes aegypti - Cell ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Proteins ; Aedes aegypti - Molecular methods
Data de publicação: 2003
