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Effects on larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin
Author(s) -
Uawithya Panapat,
Tuntitippawan Tipparat,
Katzenmeier Gerd,
Angsuthanasombat Chanan,
Panyim Sakol
Publication year - 1998
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549800201872
Subject(s) - bacillus thuringiensis , proline , helix (gastropod) , biology , mutant , mutagenesis , biochemistry , toxin , site directed mutagenesis , toxicity , amino acid , escherichia coli , chemistry , bacteria , genetics , gene , ecology , organic chemistry , snail
The possible role of α‐helices 3 and 4 in toxicity of the dipteran‐active Bacillus thuringiensis Cry4B δ‐endotoxin was investigated by employing proline substitutions via site‐directed mutagenesis. Similar to the wild‐type Cry4B, the mutant toxins were over‐expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito‐larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N‐terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild‐type toxin was significantly reduced by two‐hour preincubation with the non‐toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.