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 α-esterase multigene family, LcαE7, encodes E3. An LcαE7 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 α-over β-naphthyl acetate as substrate. Its preference ( k cat/ K m) for a range of carboxylester substrates is α-naphthyl butyrate>α-naphthyl propionate>α-naphthyl acetate>methylthiobutyrate> p-nitrophenyl acetate. The enzyme is potently inhibited by OPs ( k i [paraoxon] = 6.3 ± 1.4 × 10 7 /M/min, k i [chlorfenvinphos] = 5.9 ± 0.6 × 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, DmαE7, 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%).