Chapter 5
A Genomics Perspective on Mutant Aliesterases and Metabolic Resistance to Organophosphates
Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: November 21, 2001 | doi: 10.1021/bk-2002-0808.ch005
1
1,2
1,3
Charles Claudianos , Erica Crone , Chris Coppin , ]Robyn Russell and John Oakeshott , 1
1 *
1
CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia Divisions of Botany and Zoology and Biochemistry and Molecular Biology, School of Life Sciences, Australian National University, Canberra, ACT 0200, Australia 2
3
There are now five insect taxa for which the molecular mechanisms underlying metabolic resistance to organophosphates have been determined. Essentially only three mechanisms have been found, all involving closely related enzymes in the carboxyl/cholinesterase multigene family. None of them generate an enzyme that is kinetically efficient in degrading a broad range of organophosphates. Paradoxically however several kinetically more efficient enzymes have been found in other eukaryotes and prokaryotes and their homologs are very likely to occur in insects. These homologs may explain several cases of esterase mediated metabolic resistance whose biochemistries are distinctfromthe five cases so far resolved at a molecular level.
90
© 2002 American Chemical Society In Agrochemical Resistance; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: November 21, 2001 | doi: 10.1021/bk-2002-0808.ch005
91 Changes in carboxylesterase activity have been implicated in many cases of metabolic resistance to organophosphate (OP) insecticides in insects and mites (/). However the nature of the change is highly variable, with comparable numbers of cases showing decreases and increases in carboxylesterase activity. In the early nineties molecular mechanisms were elucidated for two systems in which greatly elevated carboxylesterase activities were associated with resistance (2, 3). Although the species, the aphid Myzus persicae and culicine mosquitoes, are not closely related, there are close similarities in the mechanisms involved. Both involve massive gene amplifications of up to 300 copies of a particular carboxylesterase gene whose product then accounts for up to 3% of the total protein of the organism. The esterases involved in the two cases are not orthologous but they are members of the same carboxyl/cholinesterase multigene family (4). In both cases the enzyme product has almost no OP degradative ability but it binds OP with great avidity and effectively sequesters it. We have been working to elucidate the molecular bases of OP resistance associated with greatly reduced carboxylesterase activities in two species of higher Diptera, the housefly Musca domestica and the sheep blowfly Lucilia cuprina (5, 6, 7). Their molecular mechanisms also prove to be remarkably similar to each other. Both species use the same amino acid substitutions in orthologous carboxylesterases to create an enzyme with some OP hydrolase activity. The enzyme involved in these species is not orthologous to either the aphid or the mosquito enzymes above, albeit it is in the same multigene family. Taken together these results suggest that there may be very few molecular options by which carboxylesterases can be used to confer OP resistance. In this paper we consider this proposition in the context of rapidly emerging data on esterase genomics. First however we outline what we have learned about the molecular mechanism of resistance in M. domestica and L. cuprina.
Mutant Aliesterases In Higher Diptera Native gel electrophoresis of M. domestica and L. cuprina reveals polymorphism for orthologous esterase isozymes (termed ALI and E3 respectively) in which variants associated with metabolic resistance to OPs appear as non-staining or only weakly staining when standard artificial substrates like methyl butyrate and naphthyl acetate are used (8, 9, 10, 11). The Mutant Aliesterase Hypothesis advanced to explain this proposes that the polymorphism reflects mutation(s) that enable the enzymes to hydrolyse phosphoester bonds like those in OPs at the expense of their
In Agrochemical Resistance; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: November 21, 2001 | doi: 10.1021/bk-2002-0808.ch005
92 ability to hydrolyse carboxylester bonds like those in the artificial substrates (9,72). Newcomb et al. (5) confirmed the Mutant Aliesterase Hypothesis by showing that the common non-staining variant of E3 in L cuprina has acquired OP hydrolytic ability as a consequence of a Gly-»Asp substitution at residue 137 in its primary sequence. Claudianos et al. (7) then showed that the equivalent ALI variant in M domestica has also acquired OP hydrolytic activity as a result of the same substitution at the same residue. Extrapolatingfromthe known three dimensional structure of the related acetylcholinesterase (AChE) enzyme, they showed that the substitution lies in the catalytic centre of the E3/ALI enzyme and indeed is so placed that the alternative Gly and Asp residues could predispose the hydrolysis of carboxylester and phosphoester linkages, respectively. The parallel between the two species also extends to a second low/nonstaining E3/ALI variant associated with a different OP resistance phenotype. The predominant Asp-137 resistance variant hydrolyses the oxon forms of a broad range of OPs but both species also have another less common variant which bestows a somewhat different spectrum of broad resistance among OPs combined with especially high levels of resistance to malathion (6, 13). The distinguishing aspect of malathion is that it has two carboxylester bonds in addition to the phosphoester linkages found in all OPs. The malathion resistance variants of E3/ALI in the two species have acquired phosphoester hydrolytic activity while improving the native enzyme's hydrolytic activity against the carboxylester linkages in malathion (albeit with varied losses of activity against the carboxylester linkages of artificial substrates such as methyl butyrate and naphthyl esters). Campbell et al. (6) showed that the malathion resistant variant in L. cuprina is due to a Trp-»Leu substitution at residue 251 in E3 and Claudianos et al. (7) then found the same substitution at the same site in ALI. Extrapolationfromthe AChE structure again localises this site to the catalytic centre, albeit it is unclear in this case how its localisation explains the biochemical differences. A Ser-251 variant of ALI was also found in M domestica. The strain carrying the variant perished before its OP biochemistry and resistance status could be tested but it may also have been associated with malathion resistance because Ser, like Leu, is a small, uncharged amino acid (6, 14). The levels of malathion resistance conferred by the Leu-251 E3/ALI mutants are around 100 fold and this accords with these enzymes' relatively efficient kinetics for malathion (specificity constants kçJK about 3.4 χ 10 M'V) (6, 13, 15). On the other hand the levels of general OP resistance conferred by both the Asp-137 and the Leu-251 enzymes rangefromnil up to 30 fold depending on the OP. This concurs with these enzymes' notably inefficient kinetics, with k
