(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 MKWWTCVVFACAAVLADDEWREVRTAQGPVRGRKHPTADMYAFYNIPYAT APTGEDKFKAPLPPPVWLEPFDAVDKHVICPQAMFQSELLGEHLVAKENC LIANVFVPNTKEKNLSVVVYFHGGAFIGGYGELLKSTQFMKANDFILVSF NYRLGIHGFLCLGTEDAPGNAGMKDQVALLRWVQKNIASFGGNPDDVTIV GSSAGSASVDLLMLSKSAKGLFHRVIPESGGNLAAFTIQRDPVENAKKHA IKLNFTNGDDIYALEKFYKTAPMEVLTAETFFDRTDSTFVFGPCVERDTG DGAFLTETPLTIFKSGNYRKLPVLYGLADMEGLLRVDFFELWKHKMNEKF SDFLPADLKFDSEEEREEVANKIKKFYFGDKPVGNDNILKYIDYFSDVIF AYPMLRAVKLHAEAGNNQVYLYEYSFVDEDSPIIPHTNVRGATHCAQSLA VYDTKNFTHLDQSPDSPALKKMKKIVREIWHNFVKTGTPVPEGSALPAWP AAGADRAPHMSLGERLELRGALLAERTRFWDDMYQRYYRDAVPPPKPPPR PRDEL
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.
Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.
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.
