Structure of an antennally-expressed carboxylesterase suggests lepidopteran odorant degrading enzymes are broadly tuned

Abstract

Insects rely on the detection of chemical cues present in the environment to guide their foraging and reproductive behaviour. As such, insects have evolved a sophisticated chemical processing system in their antennae comprised of several types of olfactory proteins. Of these proteins, odorant degrading enzymes are responsible for metabolising the chemical cues within the antennae, thereby maintaining olfactory system function. Members of the carboxyl/cholinesterase gene family are known to degrade odorant molecules with acetate-ester moieties that function as host recognition cues or sex pheromones, however, their specificity for these compounds remains unclear. Here, we evaluate expression levels of this gene family in the light-brown apple moth, Epiphyas postvittana, via RNAseq and identify putative odorant degrading enzymes. We then solve the apo-structure for EposCCE24 by X-ray crystallography to a resolution of 2.43 Å and infer substrate specificity based on structural characteristics of the enzyme's binding pocket. The specificity of EposCCE24 was validated by testing its ability to degrade biologically relevant and non-relevant sex pheromone components and plant volatiles using GC-MS. We found that EposCCE24 is neither capable of discriminating between linear acetate-ester odorant molecules of varying chain length, nor between molecules with varying double bond positions. EposCCE24 efficiently degraded both plant volatiles and sex pheromone components containing acetate-ester functional groups, confirming its role as a broadly-tuned odorant degrading enzyme in the moth olfactory organ.

Keywords: Carboxyl/cholinesterase; Crystal structure; Epiphyas postvittana; GC-MS; Odorant degrading enzyme; RNAseq.

Fig. 1

Carboxyl/cholinesterase (CCE) gene expression in Epiphyas postvittana antennae and bodies as determined by RNASeq analyses. A) Normalised readcounts of 41 EposCCE gene transcripts in antennae and bodies. The expression levels of EposCCEs were normalised between six biological replicates (three male and three female, combined) for each tissue type. Note: EposCCEs 3, 9, 11 and 13 are not expressed (normalised readcount >1) in antennae or bodies. B) Relative expression (antennae:body) of EposCCE transcripts present in both antennae and bodies. Note: EposCCEs 20 and 40 are excluded from Fig. 1B due to their lack of expression in antennal tissue samples. Blue or red font indicates predicted extracellular enzymes based on the presence of N-terminal signal peptides (SignalP-6.0).

Fig. 2

Carboxyl/cholinesterase (CCE) gene expression in male and female Epiphyas postvittana antennae as determined by RNASeq analyses. A) Normalised readcounts of 41 EposCCE transcripts in male and female antennae. The expression levels of EposCCEs were normalised between three biological replicates for each tissue type (male or female antennae). Note: EposCCEs 3, 9, 11, 13, 20 and 40 are not expressed (normalised readcount >1) in male or female antennae. B) Relative expression (male:female) of EposCCE transcripts present in both male and female antennae. Note: EposCCEs 5, 8, 10, 12 and 41 are excluded from Fig. 2B due to their lack of expression in male or female antennal tissue samples. Blue or red font indicates predicted extracellular enzymes based on the presence of N-terminal signal peptides (SignalP-6.0).

Fig. 3

Lepidopteran carboxyl/cholinesterases with validated antennal expression. Blue or red font indicates predicted extracellular enzymes based on the presence of N-terminal signal peptides (SignalP-6.0). Epiphyas postvittana (Epos) genes are shown in bold, large font, and Bombyx mori (Bmor), Spodoptera exigua (Sexi), Spodoptera littoralis (Slit), Spodoptera litura (Slitu), Antherea polyphemus (Apol), Sesamia nonagrioides (Snon) and Mamestra brassicae (Mbra) genes are shown in regular, small font. Asterisks indicate genes that have been functionally tested for their ability to degrade sex pheromones and/or plant volatiles. Node values represent bootstrap values based on 1000 replicates.

Fig. 4

Crystal structure of EposCCE24 (in green), superimposed with the structure of DmelEst6 (in orange, PDB code 5THM).

Fig. 5

Comparisons of the internal binding cavities of EposCCE24 (A) and DmelEst6 (B). Polar atoms are shown in red (oxygen) and blue (nitrogen). Key residues lining the binding cavities are shown in stick mode. The white arrows indicate the entrances of the cavities, while the white star indicates the position of the subsite in DmelEst6 (absent in EposCCE24).

Fig. 6

Docking positions of (A) Z11–14:OAc, (B) E11–14:OAc, and (C) 14:OAc inside the binding cavity of EposCCE24. Compounds are shown in stick mode, with carbon atoms in orange (Z11–14:OAc), green (E11–14:OAc) or teal (14:OAc) and oxygen atoms in red. EposCCE24 carbon atoms are shown in green, while polar atoms are shown in blue (nitrogen) and red (oxygen).

Fig. 7

Degradation of plant volatiles and lepidopteran sex pheromone compounds by recombinant EposCCE24 as determined by GC–MS analyses. A) results from screening experiments, and B) results from kinetic experiments using the behaviourally active compounds, E11–14:OAc and Z11–14:OAc. Results represent the mean (+/- SEM) result from three biological replicates. Orange bars indicate compounds known to elicit electrophysiological responses in E. postvittana antennae.