Gene_locus Report for: luccu-E3aest7

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase alphaE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp alphaE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase (LcalphaE7) as it traverses all steps in its catalytic cycle. LcalphaE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity.

Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the alphaE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcalphaE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcalphaE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase alphaE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp alphaE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase (LcalphaE7) as it traverses all steps in its catalytic cycle. LcalphaE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity.

Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the alphaE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcalphaE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcalphaE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.

The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in alpha-esterase 7 (LcalphaE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcalphaE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization.

Insect carboxylesterases from the alphaEsterase gene cluster, such as alphaE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcalphaE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of alpha-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcalphaE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical alpha/beta-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal alpha-helix that serves as a membrane anchor. Soaking of LcalphaE7 crystals in OPs led to the capture of a crystallographic snapshot of LcalphaE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcalphaE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants ( approximately 10(6) M(-1) s(-1)) indicative of a natural substrate.

Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket.

We previously showed that wild-type E3 carboxylesterase of Lucilia cuprina has high activity against Type 1 pyrethroids but much less for the bulkier, alpha-cyano containing Type 2 pyrethroids. Both Types have at least two optical centres and, at least for the Type 1 compounds, we found that wild-type E3 strongly prefers the less insecticidal configurations of the acyl group. However, substitutions to smaller residues at two sites in the acyl pocket of the enzyme substantially increased overall activity, particularly for the more insecticidal isomers. Here we extend these analyses to Type 2 pyrethroids by using fluorogenic analogs of all the diastereomers of cypermethrin and fenvalerate. Wild-type E3 hydrolysed some of these appreciably, but, again, not those corresponding to the most insecticidal isomers. Mutations in the leaving group pocket or oxyanion hole were again generally neutral or deleterious. However, the two sets of mutants in the acyl pocket again improved activity for the more insecticidal acyl group arrangements as well as for the more insecticidal configuration of the cyano moiety on the leaving group. The activities of the best mutant enzyme against the analogs of the most insecticidal isomers of cypermethrin and fenvalerate were more than ten and a hundred fold higher, respectively, than those of wild-type. The implications for resistance development are discussed.

The cloned genes encoding carboxylesterase E3 in the blowfly Lucilia cuprina and its orthologue in Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance the enzyme's ability to hydrolyse insecticides. Previous in vitro mutagenesis and expression of these modifications (G137D, in the oxyanion hole and W251L, in the acyl pocket) have confirmed their functional significance. We have systematically substituted these and nearby amino acids by others expected to affect the hydrolysis of pyrethroid insecticides. Most mutations of G137 markedly decreased pyrethroid hydrolysis. W251L was the most effective of five substitutions at this position. It increased activity with trans permethrin 10-fold, and the more insecticidal cis permethrin >130-fold, thereby decreasing the trans:cis hydrolysis ratio to only 2, compared with >25 in the wild-type enzyme. Other mutations near the bottom of the catalytic cleft generally enhanced pyrethroid hydrolysis, the most effective being F309L, also in the presumptive acyl binding pocket, which enhanced trans permethrin hydrolysis even more than W251L. In these assays with racemic 1RS cis and 1RS trans permethrin, two phases were apparent, one being much faster suggesting preferential hydrolysis of one enantiomer in each pair as found previously with other esterases. Complementary assays with individual enantiomers of deltamethrin and the dibromo analogue of cis permethrin showed that the wild type and most mutants showed a marked preference for the least insecticidal 1S configuration, but this was reversed by the F309L substitution. The W251L/F309L double mutant was best overall in hydrolysing the most insecticidal 1R cis isomers. The results are discussed in relation to likely steric effects on enzyme-substrate interactions, cross-resistance between pyrethroids and malathion, and the potential for bioremediation of pyrethroid residues.

Sequences of the esterase gene alpha E7 were compared across 41 isogenic (IV) strains of the sheep blowfly, Lucilia cuprina, and one strain of the sibling species, L. sericata. The 1.2-kb region sequenced includes sites of two insecticide resistance mutations. Gly137Asp confers resistance to organophosphorus insecticides (OPs), particularly preferring diethyl OPs such as diazinon, while Trp251Leu prefers dimethyl OPs, and particularly malathion, with the additional presence of carboxylester moieties. We found that there are just eight haplotypes among the 41 chromosomes studied: two Gly137Asp containing haplotypes, two Trp251Leu containing haplotypes, and four susceptible haplotypes, including the L. sericata sequence. While phylogenetic analysis of these haplotypes suggests that the Asp137 and Leu251 mutations each arose at least twice, evidence for recombination was detected across the region, therefore single origins for these resistance mutations cannot be ruled out. Levels of linkage disequilibrium in the data are high and significant hitchhiking is indicated by Fay and Wu' s H test but not the Tajima test. A test of haplotype diversity indicates a paucity of diversity compared with neutral expectations. Both these results are consistent with a very recent selective sweep at the Lc alphaE7 locus. Interestingly, gene duplications of three different combinations of OP resistant haplotypes were identified in seven of the isogenic (IV) strains. All three types of duplication involve an Asp137 and a Trp251 haplotype. To examine whether more haplotypes existed before the hypothesised selective sweep, fragments of alpha E7 surrounding the resistance mutations were amplified from pinned material dating back to before OPs were used. Four new sequence haplotypes, not sampled in the survey of extant haplotypes, were obtained that are all associated with susceptibility. This is suggestive of a higher historical level of susceptible allelic diversity at this locus.

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.

Two types of organophosphorus (OP) insecticide resistance are associated with reduced 'ali-esterase' (E3 isozyme) activity in Lucilia cuprina. The 'diazinon' resistance type shows generally greater resistance for diethyl than dimethyl OPs but no resistance to malathion. The 'malathion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally high malathion resistance (600 x susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malathion (malathion carboxylesterase, MCE). E3 variants from diazinon resistant strains have previously been shown to have a Gly(137) --> Asp substitution that structural modelling predicts is only about 4.6 Angstrom 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 variants of E3 and an OP susceptible variant in a baculovirus system and compared the kinetics of their products. We find that both resistance substitutions reduce ali-esterase activity and enhance OP hydrolase activity. Furthermore the Gly(137) --, Asp substitution enhances OP hydrolase activity for a diethyl OP substrate (chlorfenvinphos) more than does the Trp(251) --> Leu substitution, which is consistent with the OP cross-resistance patterns. Trp(251) --> Leu also reduces the K-m for carboxylester hydrolysis of malathion about 10-fold to 21 mu M, which is consistent with increased RICE activity in malathion resistant strains. We then present a model in which the malathion carboxylesterase activity of the E3-Leu(251) enzyme is enhanced in vivo by its OP hydrolase activity. The latter activity enables it to reactivate after phosphorylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude that the two types of resistance can be explained by kinetic changes caused by the two allelic substitutions in the E3 enzyme

Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis ("ali-esterase") in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OP's have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

PCR primers designed from the alpha-esterase gene cluster of Drosophila melanogaster have been used to isolate fragments from eight esterase genes in the Australian sheep blowfly, Lucilia cuprina. Phylogenetic analysis suggests that three are homologues of the alpha E7, alpha E8 and alpha E9 genes of the alpha-esterase cluster of D. melanogaster. A further three are also probably alpha-esterases, whereas the remaining two more closely resemble beta-esterases. Transcripts for five of the alpha-esterase genes were detected by PCR in adult midgut, consistent with a role for these enzymes in digestion and/or detoxification. Based on the tissue distribution of these transcripts, Lc alpha E7 may possibly encode the esterase, E3, which is involved in organophosphate resistance.