Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis

Promdonkoy, Boonhiang; Pathaichindachote, Wanwarang; Krittanai, Chartchai; Audtho, Mongkon; Chewawiwat, Namchai; Panyim, Sakol

doi:10.1016/j.bbrc.2004.03.102

Cited by 8 publications

(5 citation statements)

References 21 publications

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“…Even though the concentration of inclusions was increased up to 32 μg ml -1 , no significant mortality was observed in those tryptophan mutations (Table 1), suggesting that tryptophan residues at positions 222 and 226 play a crucial role in larvicidal activity. Previous studies demonstrated the importance of tryptophan residues in the structural folding and biological activity of B. thuringiensis Cyt2Aa2, Cry1Ac and Cry1Ab toxins (19). The considerably reduced toxicity of W222L and W226L mutants, however, was not associated with the structural alterations as described earlier.…”

Section: W222 and W226 Of Bina Play A Crucial Role In Larvicidal Actimentioning

confidence: 51%

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Kunthic

Promdonkoy

Srikhirin³

et al. 2011

BMB Reports

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Bacillus sphaericus produces mosquito-larvicidal binary toxin composed of BinA and BinB. While BinB is expected to bind to a specific receptor on the cell membrane, BinA interacts to BinB or BinB receptor complex and translocates into the cytosol to exert its activity via unknown mechanism. To investigate functional roles of aromatic cluster in BinA, amino acids at positions Y213, Y214, Y215, W222 and W226 were substituted by leucine. All mutant proteins were highly produced and their secondary structures were not affected by these substitutions. All mutants are able to insert into lipid monolayers as observed by Langmuir-Blodgett trough and could permeabilize the liposomes in a similar manner as the wild type. However, mosquito-larvicidal activity was abolished for W222L and W226L mutants suggesting that tryptophan residues at both positions play an important role in the toxicity of BinA, possibly involved in the cytopathological process after toxin entry into the cells. [BMB reports 2011; 44(10): 674-679]

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Section: W222 and W226 Of Bina Play A Crucial Role In Larvicidal Actimentioning

confidence: 51%

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Kunthic

Promdonkoy

Srikhirin³

et al. 2011

BMB Reports

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“…The relative importance of different charged (37), hydrophobic (3) amino acids for Cyt1Aa activity and tryptophans for Cyt2Aa2 activity (30) has been studied systematically by sitedirected mutagenesis. Knocking down activities of these proteins by amino acid substitutions revealed rather easily the significance of the replaced amino acids in Cyt function.…”

Section: Discussionmentioning

confidence: 99%

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame ( cyt1Ca ) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

2005

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Insecticidal crystal proteins of. This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3 end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.

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“…Toxin activity is usually lost or severely reduced if mutation affects these important steps. Evidences have been previously reported such as in Cyt2Aa2-W132F and W154F (20) and some truncated forms of Cyt2Aa2 (21). These mutants were unable to be solubilized in carbonate buffer and showed no larvicidal and hemolytic activities.…”

Section: Resultsmentioning

confidence: 71%

Isoleucine at position 150 of Cyt2Aa toxin from Bacillus thuringiensis plays an important role during membrane binding and oligomerization

Pathaichindachote

Rungrod²,

Audtho³

et al. 2013

BMB Reports

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Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin. [BMB Reports 2013; 46(3): 175-180]

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Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis

Cited by 8 publications

References 21 publications

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame ( cyt1Ca ) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Isoleucine at position 150 of Cyt2Aa toxin from Bacillus thuringiensis plays an important role during membrane binding and oligomerization

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