Abstract
BackgroundAcetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization.ResultsTo create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cβ of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50°C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies.ConclusionAddition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.
Highlights
Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides
Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase, which catalyzes the hydrolysis of acetylcholine
This enzyme is the target of organophosphate and carbamate insecticides which phosphorylate or carbamoylate the serine of the active site blocking the hydrolysis of the neurotransmitter acetylcholine
Summary
Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase, which catalyzes the hydrolysis of acetylcholine. This enzyme is the target of organophosphate and carbamate insecticides which phosphorylate or carbamoylate the serine of the active site blocking the hydrolysis of the neurotransmitter acetylcholine. The post-synaptic membrane remains depolarized and synaptic transmission cannot take place so the insect dies. These compounds are used to control proliferation of various agricultural pests: insects, acari and nematodes. Insecticide residues can be detected with biosensors using AChE as biological element to detect low levels of contaminants in crops, soil, water or food samples [1,2]
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