Kinetic efficiency of mutant carboxylesterases implicated in organophosphate insecticide resistance A.L. Devonshire, a,1 R. Heidari, a,b,2 K.L. Bell, a,3 P.M. Campbell, a B.E. Campbell, a W.A. Odgers, a,4 J.G. Oakeshott, a and R.J. Russell a, * a CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia b School of Agriculture, Charles Sturt University, P.O. Box 588, Wagga Wagga, NSW 2678, Australia Received 2 April 2002; accepted 24 November 2002 Abstract 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 Dro- sophila 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. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Kinetics; Malathion carboxylesterase; Organophosphorus hydrolase; Insecticide resistance 1. Introduction The hydrolytic degradation of organophos- phorus (OP) insecticides has long been implicated in the development of resistance to these com- pounds in treated insect populations [1]. One of the earliest such mechanisms proposed was the Ômutant aliesteraseÕ in houseflies (Musca domes- tica), so-called because the flies lost the ability to hydrolyse certain aliphatic esters as they gained Pesticide Biochemistry and Physiology 76 (2003) 1–13 www.elsevier.com/locate/ypest * Corresponding author. Fax: +61-2-6246-4173. E-mail address: Robyn.Russell@csiro.au (R.J. Russell). 1 Seconded to CSIRO Entomology from IACR-Rotham- sted; Harpenden, Herts, AL5 2JQ, UK. 2 Present Address: Research School of Chemistry, The AustralianNationalUniversity,Canberra,ACT2601,Australia. 3 Present address: Department of Zoology and Entomology, University of Queensland, St. Lucia, Qld. 4072, Australia. 4 Present address: Biosecurity Australia, AFFA, GPO Box 858, Canberra, ACT 2601, Australia. 0048-3575/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0048-3575(03)00054-3