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# Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of Apis mellifera

Mathieu Schwartz, Valentin Boichot, Mariam Muradova, Pablo Fournier, Patrick Senet, Adrien Nicolaï, Francis Canon, Frederic Lirussi, Ruben Ladeira, Martine Maibeche, et al.

► To cite this version:

Mathieu Schwartz, Valentin Boichot, Mariam Muradova, Pablo Fournier, Patrick Senet, et al. Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of Apis mellifera. FEBS Letters, 2023, pp.3038-3048. 10.1002/1873-3468.14770. hal-04329230

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# Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of *Apis mellifera*

Mathieu Schwartz¹ ID, Valentin Boichot¹, Mariam Muradova¹,², Pablo Fournier³, Patrick Senet⁴,
Adrien Nicolai⁴, Francis Canon¹, Frederic Lirussi⁵, Ruben Ladeira⁵, Martine Maibeche⁶,
Thomas Chertemps⁶, Emmanuel Aubert⁷, Claude Didierjean⁷ ID and Fabrice Neiers¹ ID

1 CSGA, Flavour Perception: Molecular Mechanisms (Flavours), Université de Bourgogne, INRAE, CNRS, Institut Agro, Dijon, France

2 International Research Center "Biotechnologies of the Third Millennium", Faculty of Biotechnologies (BioTech), ITMO University, Saint-Petersburg, Russia

3 Independent Researcher, Charlotte, NC, USA

4 Laboratoire Interdisciplinaire Carnot de Bourgogne, UMR 6303 CNRS, Université de Bourgogne Franche-Comté, Dijon, France

5 Plateforme PACE, Laboratoire de Pharmacologie-Toxicologie, Bioinformatique & Big Data Au Service de La Santé 2B2S, UFR Santé, Université de Franche-Comté, INSERM U1231, Centre Hospitalier Universitaire, Besançon, France

6 Institut d'Ecologie et des Sciences de l'Environnement de Paris, Sorbonne Université, INRAE, CNRS, IRD, UPEC, Paris, France

7 Université de Lorraine, CNRS, CRM2, Nancy, France

## Correspondence

F. Neiers and M. Schwartz, CSGA, Flavour
Perception: Molecular Mechanisms
(Flavours), Université de Bourgogne, INRAE,
CNRS, Institut Agro, 7 boulvard Jeanne
d'Arc, Dijon 21000, France
Tel: +33 380 6932 02
E-mail: fabrice.neiers@u-bourgogne.fr (FN);
mathieu.schwartz@inrae.fr (MS)

(Received 15 September 2023, revised 4
October 2023, accepted 9 October 2023)

doi:10.1002/1873-3468.14770

Edited by Dietmar Manstein

Glutathione transferases (GST) are detoxification enzymes that conjugate glutathione to a wide array of molecules. In the honey bee *Apis mellifera*, AmGSTD1 is the sole member of the delta class of GSTs, with expression in antennae. Here, we structurally and biochemically characterized AmGSTD1 to elucidate its function. We showed that AmGSTD1 can efficiently catalyse the glutathione conjugation of classical GST substrates. Additionally, AmGSTD1 exhibits binding properties with a range of odorant compounds. AmGSTD1 has a peculiar interface with a structural motif we propose to call ‘sulfur sandwich’. This motif consists of a cysteine disulfide bridge sandwiched between the sulfur atoms of two methionine residues and is stabilized by CH...S hydrogen bonds and S...S sigma-hole interactions. Thermal stability studies confirmed that this motif is important for AmGSTD1 stability and, thus, could facilitate its functions in olfaction.

**Keywords:** antennae; *Apis mellifera* GSTD1; glutathione transferase; odorant; stability; sulfur sandwich

Honey bees, notably *Apis mellifera*, hold significant eco-
logical and economic importance due to their pivotal
roles in pollinating crops, flowers and fruit trees [1].
Over the past decade, global declines in bee and pollina-
tor populations have been observed, resulting from a
confluence of multiple stressors encompassing biological,

environmental and chemical factors [2]. This decline
provides a pressing impetus to investigate and compre-
hend the interactions between honey bees and their
environment.

In the realm of highly social insects such as honey
bees, chemical communication assumes a vital role in

## Abbreviations

AmGSTD1, *Apis mellifera* GST Delta 1; AmGSTD1C127S, *Apis mellifera* GST Delta 1 in which the cysteine at position 127 was changed to a serine; AmGSTD1M126L, *Drosophila melanogaster* GST Delta 1 in which the methionine at position 126 was changed to a leucine; CD, circular dichroism; CDNB, 1-chloro-2,4-dinitrobenzene; DTT, dithiothreitol; GSH, glutathione; GST, glutathione transferase; ITC, isothiocyanate.
activities such as colony maintenance, developmental
processes, regulation of behaviour, caste transitions and
defensive behaviours [3]. This mode of communication
hinges on distinct anatomical systems responsible for
sensing volatile and nonvolatile chemicals—namely the
olfactory and gustatory systems. Antennae, the principal
olfactory organ of insects, consist of three main seg-
ments: the scape, pedicels and flagellum. These
segments, particularly the flagellum in hymenopterans,
are adorned with hair-like sensilla within which recep-
tors, such as odorant receptors, detect a wide array of
sensory modalities, including odours, CO2 and water.
Chemical detection in insects entails membrane recep-
tors situated on receptor neurons, supported by a
diverse range of proteins. These proteins, including
enzymes involved in metabolizing odorant molecules
and odorant-binding proteins, are pivotal for transpor-
tation, safeguarding against odorant toxicity and modu-
lating perception [4]. Prominent enzyme superfamilies
characterized in the insect antennae comprise cyto-
chrome P450 monooxygenases, glutathione transferases
(GSTs), UDP-glycosyltransferase and carboxylesterases,
which could help to protect these sensitive organs where
neurons are directly exposed to xenobiotics, while other
players, such as glutathione peroxidases, contribute to
oxidative stress resistance [5–7].

Glutathione transferases (EC 2.5.1.18) are part of a
ubiquitous protein superfamily present across all cellu-
lar organisms, including bacteria, plants, insects and
humans [8]. Based on structural similarities and the
genes encoding them, insect GSTs are categorized [9]
into six canonical classes: Delta, Epsilon, Omega,
Sigma, Theta and Zeta [10]. Delta is observed widely
in arthropods [11], and Epsilon is probably exclusive
to insects [10]. These enzymes are involved in various
biological functions, through a range of GST activities,
encompassing the covalent conjugation of glutathione
(GSH) to hydrophobic compounds with electrophilic
centres, rendering products more water-soluble [12].
Additionally, GSTs participate in noncovalent ligand
binding, peroxide deactivation, catalytic isomerization
and ester hydrolysis, among other processes [13–15].
For instance, DmGSTE14, discovered in Drosophila,
plays a pivotal role in hormone production *via* isomer-
ization, and its absence results in larval developmental
arrest [15–17].

Glutathione transferases were shown to be expressed
in the antennae of various orders of insect species [4],
such as the dipteran *Drosophila melanogaster* [18,19], as
well as in antennaed lepidopteran species [20–28] or
coleopteran antennae [29,30]. Insect antennae GSTs
serve dual roles in both safeguarding chemosensory
organs [31–34] and influencing compound detection in

insects [26,28,35-37]. Structural studies have been car-
ried out on members of the two main classes of GSTs in
insects, the epsilon and delta classes. They exhibit the
canonical GST dimer with the active site harbouring the
glutathione binding site hosted by the thioredoxin N-
terminal domain and the hydrophobic binding site
located on the helical C-terminal domain. Additionally,
specific features distinguish the delta from the epsilon in
the dimer interface, namely a clasp motif in the delta
[38] and a wafer motif in the epsilon [39].

In *A. mellifera*, 10 functional GST-encoding genes
have been identified, a significantly smaller number than
the 38 GST-encoding genes found in *D. melanogaster*
(fruit fly) and the 31 genes in *Anopheles gambiae* (mos-
quito). Focusing on insect-specific GST classes,
*A. mellifera* lacks any epsilon class enzymes and retains
only one member of the delta class (AmGSTD1), in con-
trast to the 11 delta and 14 epsilon class members in
*D. melanogaster* and 12 delta and 8 epsilon class mem-
bers in *A. gambiae* [4,40]. Notably, the AmGSTD1 is
prominently expressed in the antennae of drones,
workers and queens [5,41]. It has been suggested that
honey bees' foraging and feeding behaviours, as well as
their eusocial organization, are responsible for the low
amounts of detoxifying genes in the genome. Bees and
plants share a mutualistic interaction, in which plants
provide nectar and pollen while receiving the benefit of
the pollination activities of honey bees. However, honey
bees are not physiologically adapted to the constant
exposure to chemicals produced by plants and humans.
This attribute, however, renders honey bees especially
sensitive to certain insecticides [42].

To elucidate the function of honey bee GSTD1 (AmGSTD1), we undertook its production, purification and biochemical and structural analysis. Concurrently, we explored the enzymatic capacity towards classical substrates as well as its capacity to interact with odorants in the context of antennal expression. An original dimerization interface was discovered, which has evolved to enhance the stability supported by the biochemical and structural analysis of two proteinic variants at the positions involved in interface stabilization.

**Materials and methods**

Cloning, expression and purification of wild-type
AmGSTD1, AmGSTD1<sup>M126L</sup> and AmGSTD1<sup>C127S</sup>
enzymes

The DNA encoding the wild-type protein sequence of
*A. mellifera* GSTD1 (NP_A0A7M7GUY7) was subjected
to codon optimization for expression in *Escherichia coli*.
Codons corresponding to the initial 26 amino acids, which extended beyond the conventional insect GST sequences and potentially indicated a signal peptide, were excised. The DNA sequences encoding the AmGSTD1<sup>M126L</sup> and AmGSTD1<sup>C127S</sup> enzyme variants were generated through substitution of the codon corresponding to a methionine at position 126 with a codon encoding a leucine and the codon corresponding to a cysteine at position 127 with a serine. The synthesized DNA sequences were procured from Genewiz (Leipzig, Germany) and subsequently subcloned and inserted into the pET22b vector (Novagen, Darmstadt, Germany) via the NdeI and SacI restriction sites. The resulting plasmids were utilized for transforming the *E. coli* BL21 star strain. Colonies isolated from an LB-ampicillin agar plate (50 mg·L<sup>-1</sup> ampicillin) were employed to inoculate a 50 mL LB-ampicillin culture (50 mg·L<sup>-1</sup> ampicillin), which was then cultivated overnight at 37 °C.

Subsequently, the 50 mL culture served to inoculate a larger 4 L LB-ampicillin medium. Protein expression was induced at an OD<sub>600nm</sub> of 0.6 through the addition of 1 mM (final concentration) isopropyl β-D-1-thiogalactopyranoside, followed by continued bacterial growth for 18 h at 37 °C. The resultant cells were harvested through centrifugation (4000 g, 20 min, 4 °C), resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 100 µM DTT and 20 mM imidazole, pH 7.4) and subjected to sonication (Vibracell; Bioblock, Pittsburgh, PA, USA) at 4 °C for disruption. Following centrifugation at 20 000 g for 45 min at 4 °C, the supernatant was loaded onto a GSTrap HP 5 mL column (Cytiva, Washington, DC, USA). The column was washed with PBS buffer, pH 7.0 and GST was eluted utilizing a 50 mM Tris-HCl buffer, pH 7.4, supplemented with 10 mM reduced GSH. Fractions containing GST were pooled, dialysed against 100 mM potassium phosphate buffer at pH 6.5 and concentrated to 10 mg·mL<sup>-1</sup> using an Amicon Ultra spin column with a molecular weight cut-off of 10 kDa (Millipore, Billerica, MA, USA). Protein purity higher than 99% was confirmed via Coomassie-stained 15% SDS/PAGE gel analysis. The wild-type and variant AmGSTD1 were dialysed against 100 mM sodium phosphate buffer, pH 6.5 before kinetic measurements and far-UV circular dichroism.

## Kinetic measurements

CDNB, GSH, isothiocyanates (ITCs) and odorants were purchased from Merck (Kenilworth, NJ, USA). All substrates except GSH were dissolved in ethanol or methanol. The final solvent concentration in the reaction system was kept at a maximum of 5% (v/v).

Specific activities were determined spectrophotometrically at 20 °C using 1 mM GSH, a variable concentration of substrate and a concentration of enzyme adapted from the enzymatic rate. A 100 mM sodium phosphate buffer, pH 6.5, was used for all assays. The K<sub>M</sub> for GSH was measured

with a variable concentration of GSH and a 1 mM concentration of CDNB. CDNB glutathionylation was monitored at 340 nm (with an extinction coefficient, ε, of 9.6 mM⁻¹·cm⁻¹). Similarly, GSH-conjugated ITCs were monitored at 274 nm for allyl-ITC (ε = 7.45 mM⁻¹·cm⁻¹); benzyl-ITC at 274 nm (ε = 9.25 mM⁻¹·cm⁻¹); butyl-ITC at 274 nm (ε = 7.75 mM⁻¹·cm⁻¹); hexyl-ITC at 274 nm (ε = 6.55 mM⁻¹·cm⁻¹); phenethyl-ITC at 274 nm (ε = 8.89 mM⁻¹·cm⁻¹); and propyl-ITC at 274 nm (ε = 8.35 mM⁻¹·cm⁻¹).

Steady-state kinetic parameters were determined under the respective assay conditions, utilizing varying substrate concentrations. Nonlinear regression was employed to fit the Michaelis–Menten and Hill equations to the data, yielding values for K<sub>M</sub>, V<sub>max</sub> and the Hill coefficient. V<sub>max</sub> values were subsequently converted into k<sub>cat</sub> values based on the molar concentration of the enzyme.

To measure the ability of AmGSTD1 to interact with odorant molecules, we used a previously published method [35,43,44]. The CDNB enzyme-based competition assay allows us to identify compounds that are conjugated or bind the enzyme without conjugation (ligandin function) to decrease enzymatic activity towards CDNB. CDNB conjugation was measured at 20 °C using 1 mM GSH and CDNB, 10 nM AmGSTD1 and two concentrations (10 and 100 µM) of the tested odorant in 100 mM sodium phosphate buffer, pH 6.5. The slope of the initial rate of the reaction was measured in triplicate and compared with the slope obtained in the same conditions but supplemented with a 10 or 100 µM concentration of the tested molecule. Stock solutions of molecules were prepared in methanol at a concentration of 100 mM. All solutions were aggregate-free and stored at -20 °C. The inhibition percentage was calculated using a previous method [35,43,44], and the values presented are the mean of three independent measurements with standard deviations.

## Far-UV circular dichroism

Far-UV circular dichroism (CD) spectra were recorded using a JASCO J-815 spectropolarimeter (Tokyo, Japan) equipped with Peltier temperature control. The proteins were prepared at a concentration of 2.5 µg·mL<sup>-1</sup> in 0.01 M phosphate buffer at pH 6.5. Using a 1-cm path-length quartz cell, the protein sample spectra were recorded with a scan speed of 50 nm·min<sup>-1</sup> between 200 and 240 nm. Spectra were averaged over three scans. For each enzyme, the spectra were consistently recorded within the same quartz cell while systematically increasing the temperature in 1 °C intervals from 30 °C to 80 °C. The values obtained at 220 nm were plotted as a function of the temperature for each enzyme. The thermal denaturation data were processed using CDPAL software (version 2.18) [45]. Data were normalized and fitted to the two-state model using the standard Autofit procedure, which allowed us to calculate the
thermal denaturation midpoint $T_m$ values for AmGSTD1WT and the two variants.

## Crystallization and diffraction data collection of wild-type AmGSTD1, AmGSTD1M126L and AmGSTD1C127S

Before crystallization assays, the wild-type and variant AmGSTD1 were dialysed against 20 mM pH 8.0 Tris-HCl and concentrated to 15 mg·mL⁻¹. Crystallization trials were performed manually at 20 °C. Wild-type AmGSTD1 was crystallized in microbatches under oil by mixing 1 µL of protein with 1 µL of solution containing 16% (w/v) PEG4000, 10% (w/v) 2-propanol, 0.1 M HEPES pH 7.5 and 0.2 M ammonium sulfate. The drops were covered with 30 µL of paraffin oil (CAS n°8042-47-5; Merck). The variant AmGSTD1M126L was crystallized in sitting drop by mixing 1 µL of protein with 1 µL of solution containing 15% (w/v) PEG4000, 0.1 M trisodium citrate pH 5.6 and 0.2 M ammonium sulfate in a drop equilibrated against a reservoir filled with 100 µL of the same solution. The variant AmGSTD1C127S was crystallized in sitting drop by mixing 1 µL of protein with 1 µL of solution containing 22% (w/v) PEG4000, 0.1 M sodium acetate and 0.2 M ammonium sulfate in a drop equilibrated against a reservoir filled with 100 µL of the same solution. All crystals appeared within a few days. Crystals were cryoprotected with 20% (w/v) glycerol and flash-frozen in liquid nitrogen before synchrotron data collection. Diffraction experiments were performed on the beamline FIP2 of synchrotron ESRF and on the beamline PROXMA1 of the synchrotron SOLEIL (Saint-Aubin, France). Wild-type AmGSTD1 crystals, AmGSTD1M126L crystals and AmGSTD1C127S crystals diffracted to 2.05, 1.75 and 1.50 Å, respectively. The datasets were indexed and integrated with xds [46] and scaled with aimless [47]. The structure of AmGSTD1 was solved by molecular replacement using the coordinates of Nilaparvata lugens GST Delta (PDB code 3WYW) [48] as a model in balbes [49]. The 3D structure was built graphically with coot [50] and refined with phenix [51]. Validation was carried out with molprobity [52]. Figures were prepared using pymol (The PyMOL Molecular Graphics System, Version 2.0; Schrödinger, LLC, New York, NY, USA). Coordinates and structure factors of AmGSTD1,

AmGSTD1M126L and AmGSTD1C127S have been deposited in the Protein Data Bank under accession codes 8Q89, 8Q8A and 8Q8B, respectively. Data and refinement statistics are summarized in Table S1.

## DFT calculations

DFT calculations (B3LYP-D3/Def2TZVPP) were performed on a model of the dimer (Met-Cys)₂ (Fig. S1) using Gaussian16 software (Pittsburgh, PA, USA); hydrogen atoms were optimized while the heavy atoms were maintained at the experimental XRD geometry. Interatomic interactions were characterized through NCI analysis [53] using mwnf software [54]; calculation results are depicted in Fig. S1. Previous studies have shown that these calculation conditions are suitable for characterizing sigma-hole interactions involving chalcogen and halogen atoms [55].

## Results and Discussion

### Wild-type AmGSTD1 enzymatic activities

AmGSTD1 wild-type was obtained in pure form following one step of affinity chromatography. To assess general enzymatic competence, we conducted experiments with well-known GST substrates, encompassing 1-chloro-2,4-dinitrobenzene (CDNB) and seven commercially available isothiocyanates (ITCs), as detailed in Table 1. Notably, AmGSTD1 exhibited elevated $k_{cat}$ value alongside low $K_M$ value for CDNB. The higher $K_M$ value for GSH, 732 µM, could account for a potential underestimation of the $k_{cat}$ value measured for CDNB, which is expected to be equivalent. Due to a strong increase in the nonenzymatic reaction, the possibility of working with a higher value of GSH is limited. Despite this limitation, the observed efficiency ($k_{cat}/K_M$) towards CDNB, at 320 s⁻¹·mm⁻¹, already surpasses the efficiency values previously documented for *D. melanogaster* Delta GSTs [56]. The efficiency of AmGSTD1 closely approaches that of 281 s⁻¹·mm⁻¹ measured for *D. melanogaster* GSTD11, the most efficient *D. melanogaster* Delta GST. Interestingly, GSTD11 appears to be the most probable orthologue

<table><caption>**Table 1.** Steady-state parameters of wild-type AmGSTD1 for CDNB, GSH and different selected ITCs. $K_M$ and $k_{cat}$ values were calculated by nonlinear regression based on the Michaelis-Menten model and Hill equation. The Michaelis-Menten model was applicable for all the tested substrates except phenethyl-ITC. The $K_M$ value for GSH was determined using 1 mM CDNB substrate. The values for the electrophilic substrates were similarly obtained with a 1 mM concentration of GSH. N.A., not applicable.</caption><thead><tr><th></th><th>CDNB</th><th>GSH</th><th>Allyl-ITC</th><th>Propyl-ITC</th><th>Butyl-ITC</th><th>Hexyl-ITC</th><th>Benzyl-ITC</th><th>Phenethyl-ITC</th><th>Sulforaphane</th></tr></thead><tbody><tr><td>k<sub>cat</sub> (min<sup>-1</sup>)</td><td>1620 ± 60</td><td>2500 ± 220</td><td>322 ± 62</td><td>262 ± 12</td><td>168 ± 3</td><td>88 ± 8</td><td>128 ± 16</td><td>40 ± 2</td><td>188 ± 38</td></tr><tr><td>K<sub>M</sub> (µM)</td><td>84 ± 14</td><td>723 ± 166</td><td>192 ± 84</td><td>183 ± 19</td><td>95 ± 7</td><td>48 ± 13</td><td>96 ± 4</td><td>17 ± 1</td><td>266 ± 108</td></tr><tr><td>Hill coefficient</td><td>N.A.</td><td>N.A.</td><td>N.A.</td><td>N.A.</td><td>N.A.</td><td>N.A.</td><td>N.A.</td><td>5 ± 1</td><td>N.A.</td></tr></tbody></table>
of AmGSTD1 among the 11 Delta GSTs found in *D. melanogaster*. Using a saturating concentration of CDNB, the *K*<sub>M</sub> for GSH was measured in the same range as *K*<sub>M</sub> values measured for *Delta D. melanogaster* GSTs. The GSH efficiency is 58 s<sup>-1</sup>·mm<sup>-1</sup> compared with 360 s<sup>-1</sup>·mm<sup>-1</sup> previously measured for the DmGSTD11 orthologous enzyme. Considering the pronounced spontaneous reactivity of ITCs with GSH, determination of the ITC *K*<sub>M</sub> value is possible only when *K*<sub>M</sub> towards ITCs is low, thereby allowing work with lower ITC concentrations. In the case of AmGSTD1, *K*<sub>M</sub> and *k*<sub>cat</sub> values were measured for all six tested ITCs due to low *K*<sub>M</sub> towards these six molecules. AmGSTD1 is highly efficient with these six substrates. Significantly, a trend emerges as the carbon chain length increases, resulting in a concomitant decrease in the Michaelis–Menten constant (*K*<sub>M</sub>). Moreover, heightened steric hindrance, which is predominantly conspicuous within aromatic ITCs, demonstrates a discernible correlation with the reduction in *K*<sub>M</sub>. In contrast, this diminution in *K*<sub>M</sub>, concurrent with the elongation of the carbon chain, corresponds to a commensurate decrement in the catalytic rate. The distinctive case of sulforaphane provides compelling evidence that the sulfoxide functional moiety yields an unfavourable effect, thereby counterbalancing the influence of a four-carbon chain and inducing a threefold elevation in *K*<sub>M</sub> relative to the four-carbon butyl-ITC reference. Phenethyl-ITC was the only tested substrate in this study that exhibited strong positive cooperativity, supported by a high value of the Hill coefficient (5 ± 1). A cooperativity mechanism was previously observed for other insect GSTs of the Delta family in *D. melanogaster* or *Anopheles dirus* [57,58].

Wild-type AmGSTD1 interact with odorants

AmGSTD1 was previously shown to be highly
expressed in *A. mellifera* antennae, suggesting its
potential role in olfaction [41]. Taking into account
its efficient catalytic activity towards classical sub-
strates, which supports the functionality of this
enzyme, 27 different odorants that are mainly found in
plants and/or have shown an activity involving the
chemosensory system were tested at two concentra-
tions (Fig. 1, Table S2). A low inhibitory effect was
observed for most of them, corresponding to their
capacity to interact with AmGSTD1. The interaction
can be due to a catalytic activity towards the tested
odorant as well as a binding without catalysis. These
interactions are not surprising due to the large range
of molecules able to interact with GSTs in a general

manner. However, only three odorants were able to
inhibit AmGSTD1 at more than 30% at a concentra-
tion of 100 µM: alpha-pinene, benzaldehyde and benzyl
alcohol, three biological relevant odorants for bees.
Benzaldehyde, an odorant molecule detected by bee's
antennae and triggering aversive behaviour, appears to
be the best binder towards the tested molecules. Previ-
ously, the closest GST orthologue of AmGSTD1 in
the sphinx moth *Manduca sexta* was shown to be
highly expressed in the olfactory sensilla and active
towards trans-2-hexenal, a plant-derived green leaf
aldehyde known to stimulate the olfactory system [28].
In *Grapholita molesta*, *Plodia interpunctella* and *Sito-
philus zeamais*, orthologues of AmGSTD1 were shown
to metabolize alcohols, esters and aldehydes [37,59,60].
Recently, the GSTD1 from *Holotrichia parallela*, iden-
tified from the antennal transcriptome, was shown to
catalyse the glutathione conjugation of various unsatu-
rated aldehyde volatiles [61]. Altogether, this suggests
a possible role of AmGSTD1 in bee olfaction acting
towards various odorants.

Wild-type AmGSTD1 crystal structure analysis

We solved the crystal structure of AmGSTD1 at
2.05 Å resolution in the P4₃₂₁₂ space group, corre-
sponding to one subunit in the asymmetric unit
(Table S1). The crystal structure of AmGSTD1 corre-
sponds to the typical GST fold with an N-terminal
thioredoxin-like α/β domain and a C-terminal bundle
of α-helices (Fig. 2A). The closest structural homo-
logues showing an RMSD below 1.00 Å, as identified
by PDBFold [62], were the structures of GST delta
proteins from *N. lugens* (PDB entry 3WYW),
*A. gambiae* (PDB entry 1PN9) and GST delta 2 from
*D. melanogaster* (PDB entry 5F0G).

By the time we conducted the study, a structure of
GST delta from *A. mellifera* in complex with glutathi-
one was released without publication in the PDB
(PDB entry 7RHP). Monomeric superposition of our
structure onto this GSH-bound form yielded an
RMSD of 0.29 Å, which shows the high structural
similarity between the two structures (Fig. 2A). This
comparison allowed us to identify the glutathione
binding site residues, as our structure lacks any ligand.
The glutathione binding site residues are Glu89 and
Ser90, which stabilize the gamma-glutamyl moiety;
Ile77, which precedes cis-Pro78 and stabilizes the main
chain of the cysteinyl moiety by its main chain; Ser34,
which interacts with the cysteinyl side chain; and
His75, which interacts by H-bond with the carboxy
terminus of glutathione (Fig. 2B). These residues are
mainly conserved in the Delta class, as mentioned
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Measure of the inhibition rates of odorant molecules towards wild-type AmGSTD1 activity. Inhibition of wild-type AmGSTD1 by 27 floral odorant molecules. The inhibition rates on the CDNB activity were calculated in the presence of 10 or 100 µm odorant (light and dark grey colours, respectively). The error bars show the standard deviation and were calculated from three independent experiments. The best inhibitors are indicated by the highest inhibition rate.</figcaption></figure>

previously [35]. Additionally, our structure is devoid of any visible residues in the electron density map in the region between Val56 and Thr76, corresponding to the alpha 2 helix, which could be a flexible region important for glutathione binding, as previously shown through molecular dynamics simulation [35]. The absence of any ligand precluded an exact identification of the hydrophobic binding site (H site) residues. However, the superimposition of AmGSTD1 structure onto the previously solved structure of *A. dirus* GST delta 4 in complex with hexyl-glutathione allowed to identify putative H site residues (Fig. S2). This H site is in close vicinity of the G site as conserved in the GST family and is mainly constituted by aromatic residues.

The biological dimer of AmGSTD1, as predicted by the PISA server [63], can be generated by using crystallographic symmetry. An in-depth analysis of the interface of AmGSTD1 was carried out. AmGSTD1 exhibits the typical motif of Delta GSTs in its interface, namely the clasp motif (Fig. 2C). This structural feature in AmGSTD1 corresponds to the aromatic residue Tyr123 from subunit A that fits between helices alpha 3 and alpha 4 from subunit B. Both Tyr123 side

chains from subunits A and B are located around the twofold axis, making this structural motif important for the stability of the dimer [38]. Additionally, AmGSTD1 shows original features for a GST Delta, detailed in the following. Cys127 forms a disulfide bridge with the same Cys residue from its neighbouring subunit, thus constituting a covalent dimer (Fig. 2C). Additionally, in the vicinity of the Cys127-Cys127 disulfide bridge, the side chains of both Met126 residues interact with the S-S bond. This close proximity of the four sulfur atoms (from the Cys and Met residues) forms an unusual and unique structural motif that we propose to call the ‘sulfur sandwich’ (Fig. 2C). The distance between the sulfur atoms of Cys127 and Met126, at 2.9 Å, is significantly less than the sum of their van der Waals radii (3.6 Å). Consequently, this proximity cannot be interpreted as a van der Waals interaction. The sulfur sandwich interaction was tentatively characterized through DFT calculations on a model dimer (Met-Cys)₂ (Fig. S1), displaying the same structure than the motif ‘Met126Cys127—Cys127Met126’ in the oxidized form of AmGSTD. This analysis revealed two interactions between the Cys sulfur atom and the side chain of Met126: an
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Crystal structures of *Apis mellifera* GSTD1. (A) Global view of the AmGSTD1 dimer in cartoon view with the N-terminal domain coloured cyan and the C-terminal domain coloured magenta. A monomeric superimposition is shown with the GSH-bound form of AmGSTD1 (PDB 7RHP) in white. GSH is coloured orange, and the sulfur sandwich motif is coloured green. The missing helix alpha 2 in PDB 8Q89 (this study) is represented as red dashed lines. (B) Glutathione binding site as present in the structure of the GSH-bound form (PDB 7RHP). (C) Focus on the sulfur sandwich with an omit mFo-DFc map contoured at 3.0 sigma around the disulfide bridge (green and cyan) linking both monomers. The clasp motif has been coloured white. (D) Superimposition between PDB 8Q89 (disulfide-bridged form, green and cyan) and PDB 7RHP (reduced form, white) and focus on the sulfur sandwich.</figcaption></figure>

$^{126}$C-H...S$^{127}$ hydrogen bond involving a methylene moiety of Met126 and an $^{126}$S...S$^{127}$ sigma-hole interaction. Although the geometry of the latter interaction is not optimal [64], it is noteworthy as it is still relatively unexplored in the field of three-dimensional protein structures [65]. This sulfur sandwich is not present in the reduced structure (PDB 7RHP), as Cys127 is oriented towards their respective monomers (Fig. 2D). The same observation is true for the Met residues in the reduced structure (Fig. 2D). The sulfur sandwich probably forms upon cysteine oxidation and formation of the disulfide bond, with the methionine residues adding σ-hole effects and thus additional stability to the covalent link.

Crystal structures and thermal stability of the variants AmGSTD1$^{M126L}$ and AmGSTD1$^{C127S}$

To test the role of the cysteine and methionine residues
in the sulfur sandwich, the two variants
AmGSTD1$^{M126L}$ and AmGSTD1$^{C127S}$ were produced
and purified, and both crystal structures were solved
(isomorphous to the wild-type) (Table S1).

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Temperature denaturation of wild-type and variants AmGSTD1 followed by the normalized fractional change at 220 nm measured from CD experiments. The data were fitted with the CDPAL software and each experiment was performed in triplicate.</figcaption></figure>

Comparison of the structures of the mutants to the
wild-type revealed no major difference (Fig. S3A,B).
For AmGSTD1<sup>C127S</sup> and the side chain of the newly
introduced Ser127 points towards the monomer with
the same conformation as Cys127 in the reduced struc-
ture (Fig. S3C). For AmGSTD1<sup>M126L</sup>, the side chain
of the newly introduced Leu126 maintains a conforma-
tion similar to that of Met126 in the wild-type
(Fig. S3D). Both Cys127 sulfur atoms are at a distance
of 2.9 Å, which could indicate a partial breaking of
the disulfide bound by X-rays during diffraction exper-
iments. To assess the contribution of the two residues
M126 and C127 to the stability of the dimer, a thermal
stability study using CD was carried out on wild-type
AmGSTD1 and both variants (Fig. 3). The wild-type-
AmGSTD1 was subjected to incubation with the
dithiothreitol reductant to achieve a reduced state.
However, due to restricted access to the cysteine
residues, confirming their reduction with 5,5'-dithiobis
(2-nitro)benzoate proved unfeasible. As a result, we
were unable to assess the thermal stability of the
reduced wild-type form. The T<sub>m</sub> values were
55.5 ± 0.6 °C, 51.5 ± 0.4 °C and 48.7 ± 0.1 °C for
wild-type AmGSTD1, AmGSTD1<sup>M126L</sup> and
AmGSTD1<sup>C127S</sup>, respectively. The mutation of Cys127
to a serine and thus the disruption of the disulfide
bridge would lead to a loss in thermal stability of
6.8 °C, while the mutation of Met126 to Leu would
lead to a loss of 4.0 °C. These results show the impor-
tance of both residues Met126 and Cys127 for main-
taining the good stability of AmGSTD1.
In conclusion, the biochemical and structural char-
acterization of AmGSTD1 reveals original features for
the sole member of this class in the honey bee. The
efficient activity rate of AmGSTD1 is compatible with
a fast metabolism of odorant molecules, which is
indispensable for liberating the olfactory receptor and
facilitating renewed detection. From an evolutionary
point of view, our results suggest that keeping only
one member of GST Delta could be a disadvantage to
metabolize exogenous molecules, but this could have
been compensated by good kinetics, as well as an
enhanced stability thanks to a unique structural fea-
ture, the sulfur sandwich.

Acknowledgements

The authors would like to thank the staff of beamlines
BM07-FIP2 at ESRF and PROXIMA1 at SOLEIL
for assistance with crystal testing and data collection.
The authors received funding for this work: FN,
Agence Nationale de la Recherche grant number NF
ANR-20-CE21-0002, MS, ANR-22-CE21-0001.

Author contributions

FN and MS conceived and supervised the study. MS, VB, MM, PF, PS, AN, FC, FL, RL, MM, TC, EA, CD and FN performed the experiments and analysed the data; FN and MS wrote the manuscript.

Peer review

The peer review history for this article is available at
https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/1873-3468.14770.

Data accessibility

The structural data that support the findings of this
study are openly available in the wwPDB at https://
doi.org/10.2210/pdb8Q89/pdb, https://doi.org/10.2210/
pdb8Q8A/pdb, https://doi.org/10.2210/pdb8Q8B/pdb.

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**Supporting information**

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Fig. S1.** Non-Covalent interactions Index (NCI) analysis of the sulfur sandwich motif.

**Fig. S2.** Superimposition of AmGSTD1 monomer onto the structure of *Anopheles dirus* GST delta 4

(AdGSTD4, PDB 3F63) in complex with hexyl-glutathione.

**Fig. S3.** Crystal structures of mutants AmGSTD1<sup>M126L</sup> and AmGSTD1<sup>C127S</sup> and superimposition with the wild type structure.

**Table S1.** X-ray diffraction and refinement statistics.

**Table S2.** Ecological context of tested odors.