Enhanced pesticide sensitivity of novel housefly acetylcholinesterases: a new tool for the detection of residual pesticide contamination.
The full-length cDNA encoding an acetylcholinesterase (AChE) was cloned and sequenced from the housefly, Musca domestica, by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that this 2,076 bp sequence encodes a mature protein of 612 amino acids (67 kDa) and a 79 residue signal peptide. The amino acid sequence shared 52.8-81.4% identity with the AChE proteins of other insects. The cDNA sequence, which lacked the signal peptide was inserted into the vector pPIC9K and then introduced into strain GS115 of the yeast Pichia pastoris. The recombinant AChE protein was then expressed in P. pastoris strain GS115 by methanol induction. Site-directed mutagenesis of the A262G, Y327F, Y327D and I374D residues, either singly or in combination, was performed by reverse PCR. These mutants improved the catalytic activity and sensitivity to the organophosphate and carbamate insecticides. Although the sensitivity of other mutants was slightly increased, the results still showed that the sensitivity of triple mutant, GDD (A262G/Y327D/I374D), enhanced remarkably as much as 16 times for methomyl, 14 times for both carbofuran and chlorpyrifos, and ten times for parathion-methyl, compared to that of the wild-type. The results strongly suggested that these residues are the key structural elements controlling AChE enzyme catalytic activity and sensitivity to inhibition by insecticides. The AChE enzyme obtained by this method could be used to detect the organophosphate and carbamate insecticide residues in fruits and vegetables, a characteristic of great potential research and industrial application.
Tan
Furong
F
Biotechnology Research Institute, Shanghai Academy of Agriculture Sciences, 2901 Beidi Road, Shanghai 201106, China.
Wang
Ligang
L
Wang
Jinbin
J
Wu
Xiao
X
Zhu
Hong
H
Jiang
Lingxi
L
Tao
Shiru
S
Zhao
Kai
K
Yang
Yan
Y
Tang
Xueming
X
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002219:Carbamates / Q000032:analysis; D002800:Cholinesterase Inhibitors / Q000032:analysis; D005506:Food Contamination / Q000032:analysis*; D006793:Houseflies / Q000201:enzymology* / Q000378:metabolism; D007305:Insecticide Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010755:Organophosphates / Q000032:analysis; D010573:Pesticide Residues / Q000032:analysis*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002219:Carbamates; D002800:Cholinesterase Inhibitors; D010755:Organophosphates; D010573:Pesticide Residues; D011994:Recombinant Proteins; D000110:Acetylcholinesterase