Acetylcholinesterase engineering for detection of insecticide residues.
To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.
Boublik
Yvan
Y
Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 31062 Toulouse, France.
Saint-Aguet
Pascale
P
Lougarre
Andrée
A
Arnaud
Muriel
M
Villatte
François
F
Estrada-Mondaca
Sandino
S
Fournier
Didier
D
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D002219:Carbamates; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D004006:Dichlorvos / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology; D007306:Insecticides / Q000494:pharmacology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
D000596:Amino Acids; D002219:Carbamates; D002800:Cholinesterase Inhibitors; D007306:Insecticides; D004006:Dichlorvos; D000110:Acetylcholinesterase