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KMID : 0380220070400020163
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 2 p.163 ~ p.171
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Structurally Conserved Aromaticity of Tyr249 and Phe264 in Helix 7 Is Important for Toxicity of the Bacillus thuringiensis Cry4Ba Toxin
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Tiewsiri Kasorn
Angsuthanasombat Chanan
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Abstract
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Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry δ- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp243, Phe246, Tyr249 and Phe264, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr249 and Phe264 still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr249 and Phe264 within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.
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KEYWORD
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Bacillus thuringiensis, Conserved aromaticity, δ-endotoxin, Larvicidal activity, Site-directed mutagenesis
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