Drosophila Melanogaster Acetylcholinesterase Research Articles

Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophilamelanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

Anticholinesterase activity and antioxidant properties of Heinsia crinita and Pterocarpus soyauxii in Drosophila melanogaster model

BackgroundPlant alkaloids have become important sources of nutraceuticals owing to their pharmacological importance especially in the management of neurodegenerative diseases such as Alzheimer’s disease. In assessing the therapeutic potentials of plant phytochemicals, the fruit fly (Drosophila melanogaster) has emerged as a very veritable tool and has been largely accepted as an alternative model in biomedical research. ObjectivesIn this study, alkaloid extracts from bush apple (Heinsia crinita (Afzel.) G. Taylor and padauk (Pterocarpus soyauxii Taub.) leaves were assessed on D. melanogaster exposed to aluminum toxicity. Materials and methodsAlkaloid extracts were prepared by solvent extraction method. Thereafter, the extracts were evaluated for their in vitro antioxidant properties, Fe2+-chelating abilities and inhibitory effects on drosophila acetylcholinesterase (AChE) activity. The samples were also characterized for their constituent alkaloids via HPLC. Thereafter, effective safe dose of the extracts were determined in D. melanogaster (Harwich strain). Subsequently, flies assaulted with AlCl3 were co-treated with the extracts (8.3 and 16.6 μg/g) for seven days, during which their survival rate was monitored. This was followed by assaying for the activities of AChE, antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione-S-transferase (GST)]. Also, the flies were assayed for levels of thiobarbituric acid reaction substance (TBARS) and reactive oxygen species (ROS). ResultsThe results revealed that both extracts showed in vitro antioxidant properties with Padauk showing significantly higher antioxidant properties in vitro. However, there was no significant difference in their in vitro AChE inhibition. In vivo, Al-induced toxicity reduced survival rate, elevated AChE, SOD and GST activities, as well as TBARS and ROS levels which were ameliorated by the extracts. It was also revealed that piperine was predominant in PA, while 1-cyclohexen-1-yl-pyrrolidine was predominant in BA. ConclusionOur data suggest that the protective abilities of these extracts against Al-induced toxicity can be primarily associated with their anticholinesterase and metal chelating abilities. Thus, these vegetables can be potential sources of nutraceuticals against aluminum toxicity and associated diseases.

HETEROLOGOUS EXPRESSION OF TWO ACETYLCHOLINESTERASES OF COLORADO POTATO BEETLE LEPTINOTARSA DECEMLINEATA IN BACTERIA ESCHERICHIA COLI AND PRODUCTION OF FORM-SPECIFIC ANTIBODIES

The Colorado potato beetle Leptinotarsa decemlineata is a widespread pest of plants of the Solanaceae family. Huge economic loss caused by L. decemlineata around the world is multiplied by its ability to develop resistance to all major insecticide classes. Previously, such resistance was found to be associated with mutations in the target enzyme LdAChE2, orthologous to Drosophila melanogaster acetylcholinesterase. However, discovery of the second form of L. decemlineata acetylcholinesterase LdAChE1 has changed this view. In order to compare the role of two acetylcholinesterase forms in the Colorado potato beetle physiology and in pest resistance to insecticides, gene copies were cloned and their heterologous expression in bacteria E. coli was followed by production of polyclonal antibodies against the recombinant proteins. Immunoblotting with produced antibodies demonstrated the absence of cross-reactivity, a lower content of LdAChE1 in the tissues of L. decemlineata adults compared with the second form, and the association of LdAChE2 with membranes. Further immunoaffinity purification of natural enzymes from the beetle tissues as well as their heterologous expression in insect cell cultures should help to evaluate the role of each form in physiology of the pest and in its resistance to insecticides.

A Second Look at the Crystal Structures of Drosophila melanogaster Acetylcholinesterase in Complex with Tacrine Derivatives Provides Insights Concerning Catalytic Intermediates and the Design of Specific Insecticides.

Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at “old” diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 σ) at covalent distance from the Cβ of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks.

Mode of action of 3-butylidene phthalide as a competent natural pesticide

In this study, the biological activities and mode of action of 3-butylidene phthalide (3-BPH) were studied. 3-BPH had a superior efficiency against microsclerotia of Macrophomina phaseolina compared to the commercial fungicide tricyclazole. The microsclerotia formation and pigmentation were inhibited at 100 μg/mL. Moreover, the fungicide exhibited in silico affinity toward trihydroxy naphthalene reductase (3HNR). Both 3-BPH and tricyclazole showed congruence in the orientation and interaction within the 3HNR active site. 3-BPH displayed a strong interaction with SER-164, TYR-178, and TYR-223, with estimated binding energy and inhibition constant of −6.78 kcal mol−1, and Ki = 12.6 μM, respectively. Furthermore, it showed in vitro and in silico inhibitory activity against Drosophila melanogaster acetylcholinesterase in a concentration-dependent manner with IC50 = 730 μg/mL. It also impaired Galleria mellonella phenol oxidase enzyme, which corresponds with the insect's immune system. Phytotoxicity of 3-BPH was evident against Lemna minor at 1000 μg /mL; nevertheless, it was nontoxic at the concentrations inhibiting M. phaseolina microsclerotia and dark pigments suggest that it may be safe for use on other plants at low doses. Further assays are wanted to develop 3-BPH as a novel crop protection compound.

Design, Synthesis and Insecticide Activity of Novel Acetylcholinesterase Inhibitors: Triazolinone and Phthalimide Heterodimers.

Based on the "cluster effect" and the structure characters of acetylcholinesterase (AChE; EC 3.1.1.7), a new series of 1,2,4-triazolin-3-one and phthalimide heterodimers were designed, synthesized, and evaluated as potent dual acetylcholinesterase inhibitors (AChEIs). Most of the synthesized compounds showed good in vitro inhibitory activities towards both Drosophila melanogaster acetylcholinesterase (DmAChE) and Musca domestica acetylcholinesterase (MdAChE). Among them, 5g was found to be the most potent anti-AChE derivate (5g, IC50 = 8.07 µM to DmAChE, IC50 = 32.24 µM to MdAChE). It was 2.31- and 1.35-fold more active than the positive control ethion (CP, IC50 = 18.62 µM to DmAChE, IC50 = 43.56 µM to MdAChE). The docking model study revealed that 5g possessed the fitted spatial structure and bound to the central pocket and peripheral site of DmAChE. Moreover, most compounds demonstrated high insecticidal activity to Lipaphis erysimi and Tetranychus cinnabarinus at the concentration of 300 mg/L.

Discovery and identification of O, O-diethyl O-(4-(5-phenyl-4, 5-dihydroisoxazol-3-yl) phenyl) phosphorothioate (XP-1408) as a novel mode of action of organophosphorus insecticides

Organophosphorus (OP) insecticides play an important role in pest control. Many OP insecticides have been removed from the market because of their high toxicity to humans. We designed and synthesized a new OP insecticide with the goal of providing a low cost, and less toxic insecticide. The mode of action of O, O-diethyl O-(4-(5-phenyl-4, 5-dihydroisoxazol-3-yl) phenyl) phosphorothioate (XP-1408) was studied in Drosophila melanogaster. Bioassays showed that XP-1408 at a concentration of 50 mg/L delayed larval development. Molecular docking into Drosophila acetylcholinesterase (AChE) and voltage-gated sodium channels suggested that XP-1408 fitted into their active sites and could be inhibitory. Whole-cell patch clamp recordings indicated that XP-1408 exhibited synergistic effects involving the inhibition of cholinergic synaptic transmission and blockage of voltage-gated potassium (Kv) channels and sodium (Nav) channels. In conclusion, the multiple actions of XP-1408 rendered it as a lead compound for formulating OP insecticides with a novel mode of action.

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster

Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3′ RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics.

Structural basis of femtomolar inhibitors for acetylcholinesterase subtype selectivity: Insights from computational simulations

Acetylcholinesterase (AChE) is a key enzyme of the cholinergic nervous system. More than one gene encodes the synaptic AChE target. As the most potent known AChE inhibitor, the syn1-TZ2PA6 isomer was recently shown to have higher affinity as a reversible organic inhibitor of acetylcholinesterase1 (AChE1) than the anti1-TZ2PA6 isomer. Opposite selectivity has been shown for acetylcholinesterase2 (AChE2). In an attempt to understand the selectivity of the syn1-TZ2PA6 and anti1-TZ2PA6 isomers for AChE1 and AChE2, six molecular dynamics (MD) simulations were carried out with mouse AChE (mAChE, type of AChE1), Torpedo californica AChE (TcAChE, type of AChE1), and Drosophila melanogaster AChE (DmAChE, type of AChE2) bound with syn1-TZ2PA6 and anti1-TZ2PA6 isomers. Within the structure of the inhibitor, the 3,8-diamino-6-phenylphenanthridinium subunit and 9-amino-1,2,3,4-tetrahydroacridine subunit, via π–π interactions, made more favorable contributions to syn1-TZ2PA6 or anti1-TZ2PA6 isomer binding in the mAChE/TcAChE enzyme than the 1,2,3-triazole subunit. Compared to AChE1, the triazole subunit had increased binding energy with AChE2 due to a greater negative charge in the active site. The binding free energy calculated using the MM/PBSA method suggests that selectivity between AChE1 and AChE2 is mainly attributed to decreased binding affinity for the inhibitor.

Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was sequenced and expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haematobia irritans (L.) and Musca domestica (L.), respectively. Structural characteristics of M. domestica, H. irritans, and Drosophila melanogaster AChEs were conserved in the S. calcitrans AChE. The recombinant enzyme was inhibited by eserine, coroxon, and paraoxon and exhibited Km values of 63.9μM for acetylthiocholine and 96.7μM for butyrylthiocholine, confirming its biochemical identity as an acetylcholinesterase (EC 3.1.1.7). These data will enable rapid identification and assay for mutations that reduce AChE sensitivity to organophosphate (OP) pesticides, potentially aiding resistance management efforts to prevent fixation of the mutations in pest populations.

Disposable Screen-Printed Electrode Coupled with Recombinant Drosophila melanogaster Acetylcholinesterase and Multiwalled Carbon Nanotubes for Rapid Detection of Pesticides

Recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE), multiwalled carbon nanotubes (MWCNTs), and Prussian blue have been combined for development of a three-electrode biosensor with more rapid responses and higher stability than in our previous study. A new disposable screen-printed electrode (SPE) was developed for rapid detection of organophosphate and carbamate pesticides. After optimization, 10 microg MWCNT and 5 microL enzyme immobilization solution consisting of 0.2% glutaraldehyde, 0.1% Nafion, 0.2% bovine serum albumin, 0.1 g/L MWCNT, and 1.5 mU R-DmAChE were fixed on each of the R-DmAChE/MWCNT SPEs. The LOD of this biosensor was 0.5 microg/L for pesticide standards of dichlorvos (DDV) and carbofuran. The performance of this biosensor was tested for vegetable and water samples at various spiked levels, and good stability and sensitivity were found. The obtained recoveries were from 82.6 to 110.5% for DDV at levels of 0.5-5 microg/L and 73.4 to 118.4% for carbofuran at 1-10 microg/L in lake and sea water samples, demonstrating that the proposed approach is an alternative means for rapid detection of pesticide residues and contaminants in food safety and environmental monitoring.

Accumulation of Tetrahedral Intermediates in Cholinesterase Catalysis: A Secondary Isotope Effect Study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.

Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 °C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85–88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities ( K i = 0.123 mM and K′ i = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme–inhibitor complex. The bimolecular inhibition rate constant k i = 2.8 × 10 4 M −1 s −1 was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.

A butterfly effect: highly insecticidal resistance caused by only a conservative residue mutated of drosophila melanogaster acetylcholinesterase

Acetylcholinesterase (AChE) and its mutation recently emerged as a significant research area, due to its resistance against organophosphate and carbamate insecticides. Residue G265, which is always a conservative residue, mutated to A265 is the most frequent mutant of AChE in Drosophila populations. However, only this mutation caused a 'butterfly effect' that gives high insecticidal resistance. Herein, the models of sensitive strain (Dm-S) and the resistance strain (Dm-R) were constructed, to give a total of 2000 ps molecular dynamics simulation and to reveal the insecticidal resistance mechanism, with implied, the active gorge of Dm-R was much less flexible than that of Dm-S. The "back door" channel was widened to accelerate the detoxication against insecticides by the conformation changing of W83 and I161. All the distances (S238-H480, S238-G150, S238-G151, Y71-M153) in Dm-R became smaller than those in Dm-S, which may deeply influence the binding between the insecticides and DmAChE.

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R- DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/ DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R- DmAChE activity were obtained. The highest production of R- DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R- DmAChE in the medium could be used for production of R- DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Identification and characterization of a cDNA encoding the acetylcholinesterase ofHaematobia irritans(L.) (Diptera: Muscidae)

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.

Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues

A novel recombinant Drosophila melanogaster acetylcholinesterase (R- DmAChE) produced in Pichia pastoris was first reported in this study. We cloned the DmAChE cDNA by reverse transcription PCR with removal of the signal for glycosylphosphatidylinositol (GPI) anchor attachment and the endogenous signal peptide coding sequence, and inserted it into P. pastoris vector pPIC9K under control of the alcohol oxidase gene AOX1 promoter (5′ AOX1). The expression cassette of AChE cDNA was then introduced into methylotrophic yeast GS115 and several recombinant strains expressing R- DmAChE were obtained. The secreted R- DmAChE showed high stability in neutral phosphate buffer at 4 °C, and its kinetic parameters were identical to those of the native DmAChE. The bimolecular rate constants of R- DmAChE to dichlorvos, aldicarb and carbaryl were ranging from three to six times higher than of native DmAChE . Within six insecticides, the R- DmAChE was more sensitive than EeAChE, NbAChE and HuAChE. For 10 widely used insecticides, the IC 50 values to the R- DmAChE were much lower than those to AChEs commonly used in China. With the R- DmAChE-based assay, samples spiked with three concentrations of pesticides caused enzymatic activity inhibition with R.S.D. of 0–13.7%. These results suggest that the R- DmAChE can be useful for detection of organophosphate and carbamate insecticide residues.

The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase

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.

A semiempirical study of acetylcholine hydrolysis catalyzed by Drosophila melanogaster acetylcholinesterase

The enzymatic mechanism of acetylcholine hydrolysis was evaluated by semiempirical molecular orbital calculations with a model constructed with the coordinates of sixteen amino acids and four water molecules from the crystallographic structure of Drosophila melanogaster acetylcholinesterase (AChE, entry 1QO9 in the Protein Data Bank). Nine proposed reaction points for the hydrolysis mechanism were obtained, including four for the acylation step and five for the deacylation step. Our results indicate that in the Michaelis complex of the acylation step, a looser interaction between the substrate and the oxyanion hole may result from an amino acid change in the acyl pocket observed in insect as compared to the vertebrate enzyme. Detailed descriptions of the reaction profile for the formation of both acylation and deacylation tetrahedral intermediates were obtained. The results indicate the occurrence of partially concerted mechanisms, with deprotonation of the nucleophiles (Ser238 in the acylation step and a water molecule in the deacylation step) by His480 facilitating the nucleophilic additions. Both processes were completed by enthalpically favorable steps, formation of choline in the acylation step and of acetic acid in the deacylation step.