(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 > Diptera: NE > Brachycera: NE > Muscomorpha: NE > Eremoneura: NE > Cyclorrhapha: NE > Schizophora: NE > Calyptratae: NE > Oestroidea: NE > Calliphoridae: NE > Luciliinae: NE > Lucilia: NE > Lucilia cuprina: NE
F115S/I199V/F368Y : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina F115S : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina F368Y : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina G303A/F368Y : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina G303A : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina I199T : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina I199V/F368Y : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina I199V/G303A/F368Y : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina I199V : The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina
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 MEFEIFFLFVYLKQQISSGASTIMARFITTSSSPTLTTSTAATAPSSSWS SNATSTATSISSHSRTSRKSRYTSSNLLNAFASLTSRSSLSLSSTSSNDL YRGFLTTLVILLRMSSVAYGITDRLIVQTTSGPVRGRAVTVQGREVHVFT GIPYAKPPVDDLRFRKPVPAEPWHGVLDATRLPATCVQERYEYFPGFSGE EIWNPNTNVSEDCLYMNIWAPAKARLRHGRGANGGEHSSKTDPDHLIHSA TPQNTTNGLPILIWIYGGGFMTGSATLDIYNADIYSVGNVSVASFQYRVG AFGFLHLSPVMPGFEEEAPGNVGLWDQALALRWLKENARAFGGNPEWMTL FGESAGSSSVNAQLASPVTRGLVKRGMMQSGTMNAPWSHMTSEKAVEIGK ALINDCNCNASLLPANPQSVMACMRAVDAKTISVQQWNSYSGILSFPSAP TIDGAFLPADPMTLMKTADMSGYDIMIGNVKDEGTYFLLYDFIDYFDKDE ATSLPRDKYLEIMNNIFNKATQAEREAIIFQYTSWEGNPGYQNQQQNGRA VGDHFFTCPTNEYAQALAERGAQVHYYYFTHRTSTSLWGEWMGVLHGDEI EYFFGQPLNTSLQYRAVERELGKRMLNSVIEFAKTGNPAVDGEEWPNFSK EDPVYYVFSTDEKTEKLQRGPLAKRCSFWNDYLPKVRSWVGSECENNSAE SAAVSIIYEKQQNLLKWVIMLTIMVTCIFQ
References
Title: The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina Chen Z, Newcomb RD, Forbes E, McKenzie J, Batterham P Ref: Insect Biochemistry & Molecular Biology, 31:805, 2001 : PubMed
Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.
Title: A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly Newcomb RD, Campbell PM, Ollis DL, Cheah E, Russell RJ, Oakeshott JG Ref: Proceedings of the National Academy of Sciences of the United States of America, 94:7464, 1997 : PubMed
Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.
Lucilia cuprina (and Lucilia sericata (Green bottle fly) (Phaenicia sericata)) alpha cluster carboxylesterase (OP resistance (LcaE7) E3