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.
Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.
Resistance to organophosphorus (OP) insecticides in Lucilia cuprina arises from two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion. These mutations are not found naturally in the orthologous EST23 enzyme in Drosophila melanogaster. We have introduced the two mutations (G137D and W251L) into cloned genes encoding E3 and EST23 from susceptible L. cuprina and D. melanogaster and expressed them in vitro with the baculovirus system. The ability of the resultant enzymes to hydrolyse the phosphate ester of diethyl and dimethyl organophosphates was studied by a novel fluorometric assay, which also provided a sensitive titration technique for the molar amount of esterase regardless of its ability to hydrolyse the fluorogenic substrate used. Malathion carboxylesterase activity was also measured. The G137D mutation markedly enhanced (>30-fold) hydrolysis of both classes of phosphate ester by E3 but only had a similar effect on the hydrolysis of dimethyl organophosphate in EST23. Introduction of the W251L mutation into either gene enhanced dimethyl (23-30-fold) more than diethyl (6-10-fold) organophosphate hydrolysis and slightly improved (2-4-fold) malathion carboxylesterase activity, but only at high substrate concentration.
