Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol.

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 µg.kg-1 for six weeks) and the acaricide thymol (250 g.kg-1 applied via Apiguard gel in-hive for four weeks), both separately and in combination. RNA-seq was used to discover individual and interactive chemical effects on larval gene expression and to uncover molecular mechanisms linked to reported adult and colony phenotypes. The separate and combined treatments had distinct gene expression profiles which represented differentially affected signaling and metabolic pathways. The molecular signature of the mixture was characterised by additive interactions in canonical stress responses associated with oxidative stress and detoxification, and non-additive interactions in secondary responses including developmental, neurological, and immune pathways. Novel emergent impacts on eye development genes correlated with long-term defects in visual learning performance as adults. This is consistent with these chemicals working through independent modes of action that combine to impact common downstream pathways, and highlights the importance of establishing mechanistic links between molecular and phenotypic responses when predicting effects of chemical mixtures on ecologically relevant population outcomes.

Catalytic improvement and evolution of atrazine chlorohydrolase.

The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its k(cat)/K(m) value compared with that of the wild-type enzyme. The reduction in K(m) observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.

The molecular basis for the neofunctionalization of the juvenile hormone esterase duplication in Drosophila.

The Drosophila melanogaster enzymes juvenile hormone esterase (DmJHE) and its duplicate, DmJHEdup, present ideal examples for studying the structural changes involved in the neofunctionalization of enzyme duplicates. DmJHE is a hormone esterase with precise regulation and highly specific activity for its substrate, juvenile hormone. DmJHEdup is an odorant degrading esterase (ODE) responsible for processing various kairomones in antennae. Our phylogenetic analysis shows that the JHE lineage predates the hemi/holometabolan split and that several duplications of JHEs have been templates for the evolution of secreted ß-esterases such as ODEs through the course of insect evolution. Our biochemical comparisons further show that DmJHE has sufficient substrate promiscuity and activity against odorant esters for a duplicate to evolve a general ODE function against a range of mid-long chain food esters, as is shown in DmJHEdup. This substrate range complements that of the only other general ODE known in this species, Esterase 6. Homology models of DmJHE and DmJHEdup enabled comparisons between each enzyme and the known structures of a lepidopteran JHE and Esterase 6. Both JHEs showed very similar active sites despite low sequence identity (30%). Both ODEs differed drastically from the JHEs and each other, explaining their complementary substrate ranges. A small number of amino acid changes are identified that may have been involved in the early stages of the neofunctionalization of DmJHEdup. Our results provide key insights into the process of neofunctionalization and the structural changes that can be involved.

The enzymatic basis for pesticide bioremediation.

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.

Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila.

Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate.

Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability.

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 αE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcαE7-4a, 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 LcαE7-4a 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.

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

Heterologous expression and biochemical characterisation of fourteen esterases from Helicoverpa armigera.

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.

Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

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.

Testing the evolvability of an insect carboxylesterase for the detoxification of synthetic pyrethroid insecticides.

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.