g0t5q2 quite different only n-term Pfam A COesterase 2 526 the c-term with NGGEGDGGNG repeat is not represented here. Metabolize beta-cypermethrin, lambda-cyhalothrin and fenvalerate. Carboxylesterase 001G
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Metazoa: NE > Eumetazoa: NE > Bilateria: NE > Protostomia: NE > Ecdysozoa: NE > Panarthropoda: NE > Arthropoda: NE > Mandibulata: NE > Pancrustacea: NE > Hexapoda: NE > Insecta: NE > Dicondylia: NE > Pterygota: NE > Neoptera: NE > Holometabola: NE > Amphiesmenoptera: NE > Lepidoptera: NE > Glossata: NE > Neolepidoptera: NE > Heteroneura: NE > Ditrysia: NE > Obtectomera: NE > Noctuoidea: NE > Noctuidae: NE > Heliothinae: NE > Helicoverpa: NE > Helicoverpa armigera: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MWWRTCVVLVCVTAVLADEEWRQVNTAQGPVRGQKHPSGHLYAFYNVPYA TAPKGIHKFKAPLPPPVRTEPFDAVNKNVVCPQYIDMDSKMPGRHLEQSE DCLIANIFVPDTNENNLPVLVYVHGGAFILGFGDMMRATQLMKSKDFIMV TFNYRLGIHGFLCLGTEDVPGNAGLKDQVALLRWVKDNIANFGGNPNKVT LAGYSAGSTSVDLLMLSKAAKGLFHRVIPESGANLAAFGMQRDPLEVAKS HAKTLQFTNVEDISALEQFYKTASMALLTEDSFLLRPDSTFLFGPCVERD TEGAFLTESPLTILKNGDYEKLPMLYGFANMEGLFRIDMFNDWKDKMNSN FAEFLPADLKFETDGAKEEVIELIKKFYFGGQPVSNDNILRYVDYFSDVP FVYPTLRSAKLQVEAGNDQVYLYEYSFVDENDNVVPHTNVRGADHVAQTV ALMDGEDESLKSQAYQNMRKIIREIWHNFIITGTPVPADSSLPAWPPARA DRSPHMRLLEKPVLQGVLLGERAQFWDDIYEKYYLDAVPPAAPNT
BACKGROUND: Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as beta-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. RESULTS: In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate alpha-naphthyl acetate. Inhibition study showed that beta-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward beta-cypermethrin with specific activities between 4.0 and 5.6 nmol L(-1) min(-1) mg(-1) protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the beta-cypermethrin molecule in the active binding pocket of the mutant enzymes. CONCLUSION: The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of beta-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. 2020 Society of Chemical Industry.
Title: Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera Bai LS, Zhao CX, Xu JJ, Feng C, Li YQ, Dong YL, Ma ZQ Ref: Pestic Biochem Physiol, 157:69, 2019 : PubMed
Carboxylesterases (CarEs) are a major class of detoxification enzymes involved in insecticide resistance in various insect species. In this study, a novel CarE 001G was isolated from the cotton bollworm Helicoverpa armigera, one of the most destructive agricultural insect pests. The open reading frame of 001G has 2244 nucleotides and putatively encodes 747 amino acid residues. The deduced CarE possessed the highly conserved catalytic triads(Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly), suggesting 001G is biologically active. The truncated 001G was successfully expressed in Escherichia coli, and the recombinant proteins were purified and tested. The enzyme kinetic assay showed the purified proteins could catalyze two model substrates, alpha-naphthyl acetate and beta-naphthyl acetate, with a kcat of 8.8 and 2.3s(-1), a Km of 9.6 and 16.2muM, respectively. The inhibition study with pyrethroid, organophosphate and neonicotinoid insecticides showed different inhibition profile against the purified CarE. The HPLC assay demonstrated that the purified proteins were able to metabolize beta-cypermethrin, lambda-cyhalothrin and fenvalerate insecticides, exhibiting respective specific activities of 1.7, 1.4 and 0.5nM/min/mg protein. However, the purified proteins were not able to metabolize the chlorpyrifos, parathion-methyl, paraoxon-ethyl and imidacloprid. The modeling and docking analyses consistently demonstrated that the pyrethroid molecule fits snugly into the catalytic pocket of the CarE 001G. Collectively, our results suggest that 001G may play a role in pyrethroids detoxification in H. armigera.
Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.
BACKGROUND: Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as beta-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. RESULTS: In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate alpha-naphthyl acetate. Inhibition study showed that beta-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward beta-cypermethrin with specific activities between 4.0 and 5.6 nmol L(-1) min(-1) mg(-1) protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the beta-cypermethrin molecule in the active binding pocket of the mutant enzymes. CONCLUSION: The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of beta-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. 2020 Society of Chemical Industry.
Title: Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera Bai LS, Zhao CX, Xu JJ, Feng C, Li YQ, Dong YL, Ma ZQ Ref: Pestic Biochem Physiol, 157:69, 2019 : PubMed
Carboxylesterases (CarEs) are a major class of detoxification enzymes involved in insecticide resistance in various insect species. In this study, a novel CarE 001G was isolated from the cotton bollworm Helicoverpa armigera, one of the most destructive agricultural insect pests. The open reading frame of 001G has 2244 nucleotides and putatively encodes 747 amino acid residues. The deduced CarE possessed the highly conserved catalytic triads(Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly), suggesting 001G is biologically active. The truncated 001G was successfully expressed in Escherichia coli, and the recombinant proteins were purified and tested. The enzyme kinetic assay showed the purified proteins could catalyze two model substrates, alpha-naphthyl acetate and beta-naphthyl acetate, with a kcat of 8.8 and 2.3s(-1), a Km of 9.6 and 16.2muM, respectively. The inhibition study with pyrethroid, organophosphate and neonicotinoid insecticides showed different inhibition profile against the purified CarE. The HPLC assay demonstrated that the purified proteins were able to metabolize beta-cypermethrin, lambda-cyhalothrin and fenvalerate insecticides, exhibiting respective specific activities of 1.7, 1.4 and 0.5nM/min/mg protein. However, the purified proteins were not able to metabolize the chlorpyrifos, parathion-methyl, paraoxon-ethyl and imidacloprid. The modeling and docking analyses consistently demonstrated that the pyrethroid molecule fits snugly into the catalytic pocket of the CarE 001G. Collectively, our results suggest that 001G may play a role in pyrethroids detoxification in H. armigera.
Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.
Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.
