title: cDNA cloning, baculovirus-expression and kinetic properties of the esterase, E3, involved in organophosphorus resistance in Lucilia cuprina.
abstract: Resistance to organophosphorus insecticides (OPs) in the sheep blowfly, Lucilia cuprina, is associated with a non-staining phenotype of the carboxylesterase isozyme, E3 (E.C. 3.1.1.1). Here, we show that a member of alpha-esterase multigene family, Lc alpha E7, encodes E3. An Lc alpha E7 cDNA has been isolated from an OP-susceptible strain and expressed in a baculovirus. The expressed product is the same as E3 in its electrophoretic mobility and preference for alpha-over beta-naphthyl acetate as substrate. Its preference (kcat/K(m)) for a range of carboxylester substrates is alpha-naphthyl butyrate > alpha-naphthyl propionate > alpha-naphthyl acetate > methylthiobutyrate > p-nitrophenyl acetate. The enzyme is potently inhibited by OPs (ki [paraoxon] = 6.3 +/- 1.4 x 10(7)/M/min, ki [chlorfenvinphos] = 5.9 +/- 0.6 x 10(7)/M/min) and exhibits a high turnover of methylthiobutyrate (1009/s), consistent with its proposed homology to the ali-esterase that is thought to mutate to confer OP resistance in Musca domestica. E3 shares 64% amino acid identity with its Drosophila melanogaster homologue, Dm alpha E7, and is also closely related to other esterases involved in OP resistance such as the B1 esterase of Culex pipiens (38%) and E4 of Myzus persicae (30%).
authors.affiliation: CSIRO, Division of Entomology, Canberra, Australia.
authors.forename: R D
authors.initials: RD
authors.lastname: Newcomb
authors.forename: P M
authors.initials: PM
authors.lastname: Campbell
authors.forename: R J
authors.initials: RJ
authors.lastname: Russell
authors.forename: J G
authors.initials: JG
authors.lastname: Oakeshott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002802:Cholinesterases / Q000737:chemistry; D018076:DNA, Complementary; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007305:Insecticide Resistance; D007306:Insecticides; D007700:Kinetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology
chemical_list: D018076:DNA, Complementary; D007306:Insecticides; D009943:Organophosphorus Compounds; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D002802:Cholinesterases