2 DIFFERENT AMINO-ACID SUBSTITUTIONS IN THE ALI-ESTERASE, E3, CONFER ALTERNATIVE TYPES OF ORGANOPHOSPHORUS INSECTICIDE RESISTANCE IN THE SHEEP BLOWFLY, LUCILIA-CUPRINA

Two types of organophosphorus (OP) insecticide resistance are associat ed with reduced 'ali-esterase' (E3 isozyme) activity in Lucilia cuprin a. The 'diazinon' resistance type shows generally greater resistance f or diethyl than dimethyl OPs but no resistance to malathion. The 'mala thion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally hi gh malathion resistance (600 x susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malat hion (malathion carboxylesterase, MCE). E3 variants from diazinon resi stant strains have previously been shown to have a Gly(137) --> Asp su bstitution that structural modelling predicts is only about 4.6 Angstr om from the gamma oxygen of the catalytic serine residue. Here we show that E3 variants from malathion resistant strains have a Trp(251) --> Leu substitution predicted to be about 4.3 Angstrom from that serine. We have expressed alleles of the gene encoding both resistance varian ts of E3 and an OP susceptible variant in a baculovirus system and com pared the kinetics of their products. We find that both resistance sub stitutions reduce ali-esterase activity and enhance OP hydrolase activ ity. Furthermore the Gly(137) --, Asp substitution enhances OP hydrola se activity for a diethyl OP substrate (chlorfenvinphos) more than doe s the Trp(251) --> Leu substitution, which is consistent with the OP c ross-resistance patterns. Trp(251) --> Leu also reduces the K-m for ca rboxylester hydrolysis of malathion about 10-fold to 21 mu M, which is consistent with increased RICE activity in malathion resistant strain s. We then present a model in which the malathion carboxylesterase act ivity of the E3-Leu(251) enzyme is enhanced in vivo by its OP hydrolas e activity. The latter activity enables it to reactivate after phospho rylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude t hat the two types of resistance can be explained by kinetic changes ca used by the two allelic substitutions in the E3 enzyme. (C) 1998 Elsev ier Science Ltd. All rights reserved.