Molecular recognition of wood polyphenols by phase II detoxification enzymes of the white rot Trametes versicolor

Abstract

Wood decay fungi have complex detoxification systems that enable them to cope with secondary metabolites produced by plants. Although the number of genes encoding for glutathione transferases is especially expanded in lignolytic fungi, little is known about their target molecules. In this study, by combining biochemical, enzymatic and structural approaches, interactions between polyphenols and six glutathione transferases from the white-rot fungus Trametes versicolor have been demonstrated. Two isoforms, named TvGSTO3S and TvGSTO6S have been deeply studied at the structural level. Each isoform shows two distinct ligand-binding sites, a narrow L-site at the dimer interface and a peculiar deep hydrophobic H-site. In TvGSTO3S, the latter appears optimized for aromatic ligand binding such as hydroxybenzophenones. Affinity crystallography revealed that this H-site retains the flavonoid dihydrowogonin from a partially purified wild-cherry extract. Besides, TvGSTO6S binds two molecules of the flavonoid naringenin in the L-site. These data suggest that TvGSTO isoforms could interact with plant polyphenols released during wood degradation.

Full text

1
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
www.nature.com/scientificreports
Molecular recognition of
wood polyphenols by phase II
detoxication enzymes of the
white rot Trametes versicolor
Mathieu Schwartz1, Thomas Perrot2, Emmanuel Aubert1, Stéphane Dumarçay3,
Frédérique Favier1, Philippe Gérardin3, Mélanie Morel-Rouhier2, Guillermo Mulliert1,
Fanny Saiag2, Claude Didierjean1 & Eric Gelhaye
2
Wood decay fungi have complex detoxication systems that enable them to cope with secondary
metabolites produced by plants. Although the number of genes encoding for glutathione transferases
is especially expanded in lignolytic fungi, little is known about their target molecules. In this study, by
combining biochemical, enzymatic and structural approaches, interactions between polyphenols and
six glutathione transferases from the white-rot fungus Trametes versicolor have been demonstrated.
Two isoforms, named TvGSTO3S and TvGSTO6S have been deeply studied at the structural level. Each
isoform shows two distinct ligand-binding sites, a narrow L-site at the dimer interface and a peculiar
deep hydrophobic H-site. In TvGSTO3S, the latter appears optimized for aromatic ligand binding such
as hydroxybenzophenones. Anity crystallography revealed that this H-site retains the avonoid
dihydrowogonin from a partially puried wild-cherry extract. Besides, TvGSTO6S binds two molecules
of the avonoid naringenin in the L-site. These data suggest that TvGSTO isoforms could interact with
plant polyphenols released during wood degradation.
e microbial degradation of wood has been extensively studied due to its importance in organic matter recy-
cling and its potential valorisation in many industrial domains. is degradation is mainly mediated by fungi
and in particular by white-rot fungi which are able to degrade and mineralize all the wood components. Indeed,
as early as in the middle of last century, this functional trait has been correlated to the ability of these fungi to
secrete extracellular enzymatic systems able to degrade wood polymers1. anks to the recent release of more
than y fungal genomes, comparative genomic approaches have conrmed this correlation2,3. Beyond these
extracellular systems, recent studies have also conrmed the importance of intracellular detoxication systems in
the fungal wood degradation process4. ese systems were thought to play essential roles in wood degradation.
ey allow fungi to (i) catabolize the oxidized compounds that result from lignin oxidation5, and (ii) cope with
wood anti-microbial compounds, such as avonoids, stilbenes or terpenes6,7. e eciency of these intracellu-
lar detoxication systems seems to be linked to the expansion of multigenic families involved in the oxidation
phase such as cytochrome P450 mono-oxygenases and in the conjugation phase such as glutathione transferases
(GSTs)6,8. Similarly, such expansions are also found in herbivorous insects, where these multigenic families play
key functions in the detoxication of plant defense chemicals and also in the evolution of metabolic resistance to
chemical insecticides9–11.
Until now, in wood-decaying fungi, comparative genomic, biochemical, structural or physiological approaches
gave only few insights into the function and specicity of these enzymes in the wood degradation process. is
lack of knowledge is mainly due to the absence of specic substrates that would allow discrimination between
the isoforms. e expansion of the GST family in these fungi mainly concerns three phylogenetic classes, named
GSTFuA, Ure2p and GST Omega12. We had suggested that the fungal-specic GSTFuA class could be involved in
the catabolism of lignin derived molecules13. A recent study conrmed that an isoform from Dichomitus squalens
1Université de Lorraine, CNRS, CRM2, Nancy, France. 2Université de Lorraine, INRA, IAM, Nancy, France. 3Université
de Lorraine, LERMAB, Nancy, France. Mathieu Schwartz and Thomas Perrot contributed equally to this work.
Correspondence and requests for materials should be addressed to C.D. (email: claude.didierjean@univ-lorraine.fr)
or E.G. (email: eric.gelhaye@univ-lorraine.fr)
Received: 5 February 2018
Accepted: 4 May 2018
Published: xx xx xxxx
OPEN
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
2
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
(Ds-GST1) selectively cleaves the β-O-4 aryl ether bond of a dimeric lignin model leading to a glutathione deriv-
ative14. Concerning the Ure2p class, it can be divided in two subclasses with distinct structural and biochemical
properties. Ure2pAs possess the classic GSH transferase activity while Ure2pBs display a deglutathionylation
activity (Ure2pB)15. Interestingly, bacterial orthologs of Ure2pB (named GST Nu) act as glutathione lyases in
breaking the β-aryl ether bond of lignin16. GSTOs are involved in detoxication pathways via deglutathionylation
reactions17 and two GSTO isoforms of Phanerochaete chrysosporium (PcGSTO3 and PcGSTO4) bind terpenes18.
In Trametes versicolor, some GSTO isoforms interact with dierent wood extractives19. ese interactions could
give insights into the chemical composition of the extracts.
To further explore this issue, we have conducted biochemical and crystallographic studies on the interactions
of six GSTOs from T. versicolor with chemical libraries. We showed that these GSTOs exhibit distinct anity pat-
terns, particularly with benzophenones and avonoids. An anity crystallography approach allowed the isolation
of a avonoid from a partially-puried wild-cherry tree extract. is ligand specic to one GSTO isoform was
characterized as dihydrowogonin using multiple approaches. All these results and the recent literature support
the conclusion that GSTs of this class interact with wood polyphenolic compounds.
Results and Discussion
TvGSTO3S interacts with hydroxybenzophenones. ermal shi assay (TSA) is a high-throughput
ligand-screening method based on the modication of protein thermal denaturation. According to a gradient of
temperature, the denaturation is followed by monitoring uorescence enhancement of a probe (SYPRO Orange)
that binds to protein hydrophobic patches upon denaturation process. is TSA method has been success-
fully used to detect interactions between proteins and libraries of molecules20. It allowed us to identify chem-
ical families of compounds that interact with TvGSTOs and prompted us to investigate more deeply the case of
TvGSTO3S with hydroxybenzophenones (HBPs), in particular by conducting a structural analysis of protein-li-
gand complexes.
First, the interactions between a chemical library of 27 compounds and six TvGSTOs were explored using
TSA (Supplementary Fig.S1). e tested compounds were chosen either for their presence in wood or their
reactivity with GSTs19. e six TvGSTOs (named TvGSTO1S to TvGSTO6S) used in this study are representa-
tives of the twelve TvGSTOs that have a catalytic serine, while four others have a cysteine instead. e obtained
results show patterns of interaction that distinguish each isoform from the others. Indeed, a few compounds
signicantly increased the stability of TvGSTO1S, 3S and 6S, i.e. they show a variation of the denaturation tem-
perature ∆Td > 5 °C. On the contrary, most chemical compounds induced a destabilizing eect on TvGSTO5S
and 2S (∆Td < 0 °C), whereas the chemical library had little impact on TvGSTO4S thermal stability. In the case
of TvGSTO3S, the compounds that increased protein stability all belong to the same chemical family, namely
hydroxybenzophenones (HBPs). Benzophenones are present in plant extracts21 and also in wood extractives, for
example in oak heartwood22.
A set of commercially available HBPs with various numbers and positions of hydroxylation on their rings
A and B (Table1) was tested by using TSA and the six TvGSTOs. It conrmed that TvGSTO3S has a signicant
anity for HBPs. For this isoform, a group of molecules (2,4-, 3,4-, 2,3,4- and 2,4,4′- HBPs) corresponding to
compounds with at least two hydroxyl groups on ring A and no more than one hydroxylation on ring B stand out
for their causing large increases in the melting temperature of TvGSTO3S. Oppositely, 2,2′-, 4,4′-, 2,2′,4,4′- HBPs
and the unsubstituted benzophenone had little or no eect on the thermal stability of TvGSTO3S. e replace-
ment of the 4-hydroxyl group of 2,4-HBP by a methoxy group nullied the observed thermal shi.
For further insights into these ndings, a structure/function relationship study focused on the analysis of
TvGSTO3S-ligand interactions was initiated. e structure of apo TvGSTO3S was determined by X-ray crys-
tallography at a resolution of 1.35 Å (Supplementary TableS1). It has a typical GST fold where the N-terminal
thioredoxin domain of one monomer cross-interacts with the C-terminal all-helical domain of the second one,
and vice-versa (Fig.1). TvGSTO3S displays the highest sequence identity with Omega GSTs, though its closest
structural homologs identied by PDBeFold23 are Tau GSTs. e resemblance between these two classes was
already discussed for a wheat Tau GST24. However, TvGSTO3S has unique features that distinguish it from pre-
viously described GSTs. ey mainly include an elongation of the loop between β3 and β4 and an additional helix
α6′ (Supplementary Fig.S2).
e N-terminal end of TvGSTO3S helix α1 harbors a serine as the catalytic residue instead of the cysteine
found in the other GSTOs structurally characterized so far17,25. It enables efficient GSH-transferase activ-
ity towards usual synthetic substrates and disables reductase activity (Supplementary TableS2). In order to
get a detailed picture of the ligand binding sites of TvGSTO3S, we determined the structures of several com-
plexes with free GSH, or some glutathionylated derivatives GS-R (glutathionyl-dinitrobenzeneGS-DNBand
glutathionyl-phenylacetophenone GS-PAP), or some of the HBPs identied by TSA (2,4-, 3,4-, 2,3,4-, 2,4,4′-
HBPs) (Supplementary TableS1). TvGSTO3S harbors a canonical GSH binding site (G-site) made up of polar
residues from the N-terminal domain which stabilizes the glutathionyl moiety (GS-) of the tested ligands (Fig.1,
Supplementary FigsS3 and S4). On the contrary, the hydrophobic binding site (H-site) which hosts the -R group
of the GS-R ligand has a singular shape relative to most GSTs. While a large open valley is usually observed in
a cle between the two domains26, TvGSTO3S exhibits a well-delineated cavity deeply inserted between helices
α4 and α6 of the C-terminal domain. e crystal structures of the dierent complexes show that this pocket is
perfectly suited to accommodate polyaromatic ligands, due to its strong hydrophobic character, while two polar
residues are found at the entrance close to the G-site (Supplementary Fig.S3). e phenylacetophenone group of
GS-PAP fully lls the cavity, on the contrary to the more polar -R group of GS-DNB that does not enter it and is
only slightly stabilized at its entrance, at the interface with the G-site (Supplementary Fig.S4).
e four HBPs (2,4-, 2,3,4-, 3,4- and 2,4,4′- HBPs) that were previously identied by TSA also bind in the
TvGSTO3S H-site (Fig.2 and Supplementary Fig.S5). Overall, their phenyl ring B (Table1) sits deep at the bottom
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
3
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
of the pocket and interacts via π-stacking with aromatic amino acid side chains. eir di- or tri-hydroxylated phe-
nyl ring A is closer to the entrance of the cavity and forms hydrogen bonds with polar side chains and water mol-
ecules. In more detail, two distinct HBP conformations are observed (i) for 2,4-, 2,3,4- and 3,4-HBPs, and (ii) for
2,4,4′- HBP, respectively (Fig.2). Both conformations are common for this family of molecules in the solid state27.
In (i), the central ketone group of HBPs is stabilized via a well conserved water molecule present in all structures,
while this interaction no longer exists in (ii). For the latter, a new hydrogen bond allows accommodation of the 4′
hydroxyl group at the bottom of the cavity. e presence of this additional 4′ hydroxyl group probably accounts
for the distinct conformation (ii) observed in the H-site. Finally, another attempt was made to get the structure
of a complex between TvGSTO3S and the compound 2,2′, 4,4′-HBP, but no electron density was observed for
this putative ligand. ese results not only correlate with TSA, but also with the binding anities of HBPs for
TvGSTO3S which were assessed from their capacity to inhibit the GSH-transferase activity towards phenethyl
isothiocyanate(PEITC). Indeed, the same four HBPs dierentiate from the others in the sense that only their Ki
values were measurable and found in the μM range (Supplementary TableS3). Altogether, our crystallographic
and enzymatic results suggest that the TvGSTO3S H-site is selective for HBPs whose ring B is totally hydrophobic
and stabilized at the bottom of the pocket or bears one hydroxyl group in para position whereas ring A is di- or
tri- hydroxylated and stabilized at the entrance of the H-site. As shown with TSA, other TvGSTO isoforms also
interact with HBPs, each with its own selectivity. e discovery of HBPs as potential ligands for TvGSTOs echoes
previous results concerning metabolization of these small molecules by the fungus. For instance, the sunscreen
agent BP3 (2-hydroxy, 4-methoxy benzophenone) is metabolized by T. versicolor into various HBPs (2,4-, 4,4′-
and 4- HBPs) by cytochrome P450 with no oxidation by extracellular laccases28 suggesting the importance of
intracellular systems to detoxify benzophenones, including phase II enzyme GSTs.
TvGSTOs interact with avonoids. In a recent paper, we suggested that GSTs from saprotrophs could
interact with avonoids, which are phenolic compounds as HBPs and present in wood extracts19. In order to
rene our results, TSA was used to test a set of commercial avonoids for their interaction with TvGSTOs. Some
of the putative complexes were further investigated by X-Ray crystallography. For the rst time in GSTs, one
structure revealed a symmetrical ligandin site (L-site) lled with a pair of interacting ligands.
As observed for HBPs, TSA shows that each TvGSTO has its own interaction prole with avonoids and that
these interactions are largely related to the number and positions of the hydroxyl groups on the aromatic rings
(Supplementary TableS4). While TvGSTO4S and 5S show variable or weak responses to avonoids, TvGSTO1S,
Molecules Structures TvGSTO1S TvGSTO2S TvGSTO3S TvGSTO4S TvGSTO5S TvGSTO6S
Benzophenone n.s. n.s. n.s. n.s. n.s. n.s.
2,2′-Dihydroxy benzophenone n.s. n.s. n.s. n.s. n.s. n.s.
2,4-Dihydroxy benzophenone 2.05 °C 2.56 °C 5.69 °C n.s. n.s. 1.63 °C
3,4-Dihydroxy benzophenone 0.98 °C n.s. 4.36 °C 2.77 °C n.s. n.s.
4,4′-Dihydroxy benzophenone n.s. n.s. 1.67 °C n.s. n.s. n.s.
2,3,4-Trihydroxy benzophenone 1.13 °C 2.92 °C 4.96 °C 1.76 °C −12.89 °C 4.15 °C
2,4,4′-Trihydroxy benzophenone 2.74 °C 2.03 °C 2.87 °C n.s. n.s. 1.28 °C
2,2′,4,4′-Tetrahydroxy benzophenone 3.74 °C 0.74 °C n.s. n.s. n.s. 3.40 °C
2-Hydroxy-4-methoxy benzophenone n.s. n.s. n.s. n.s. n.s. n.s.
Table 1. Summary of the results obtained for TvGSTOs with benzophenone compounds by thermal-shi
assays. A ∆Td value is only given if the denaturation temperature is signicantly modied in the presence of
compounds, with respect to incubation with DMSO only. “n.s.” means that the denaturation temperature has
not changed signicantly.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
4
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
2S, 3S and 6S mainly show positive shis of their denaturation temperature. For instance, all molecules but cate-
chin aected TvGSTO6S. Interestingly, these avonoids were recently detected by mass spectrometry in T. versi-
color fructications. eir presence might arise from trees or soils on which the fungi grow29.
e crystal structure of TvGSTO6S was solved at 1.45 Å resolution (Supplementary TableS1). As expected,
it reveals the same overall fold than TvGSTO3S (rmsd 0.78 Å, 183 aligned Cα per monomer). While G-sites are
made of similar polar residues, dierences are observed at the H-site and at the interface between monomers
(Fig.3). e lack of a structure with a ligand that binds in TvGSTO6S H-site precluded its ne description.
However, when compared with the equivalent region of TvGSTO3S, signicant dierences are readily apparent
in TvGSTO6S. Most of the residues that line the benzophenone binding site of TvGSTO3S are dierent. In addi-
tion, the extension of the C-terminal α-helix by two extra turns leads to an open and shallow H-site, contrary to
the buried H-site in TvGSTO3S covered and closed by the loop corresponding to the extra turns. ere are also
slight dierences between the dimer-interfaces of both isoforms. Most residues at the interface are conserved,
except for a few ones situated in helix α4 (Fig.3). e larger residues of TvGSTO6S (L112 and T115 instead of
T110 and A113 in TvGSTO3S) and the loss of the inter-monomer interaction between Y118 (replaced by E120 in
TvGSTO6S) and E80 present in TvGSTO3S probably account for a more open dimer in TvGSTO6S. Its interface
residues form a pocket centered on the two-fold axis of the dimer (rectangular section of 8 × 6 Å2), larger than the
equivalent region found in TvGSTO3S (rectangular section of 7 × 4 Å2, Supplementary Fig.S6). is region hosts
a third binding site named the ligandin site (L-site) in the human GST Omega 1 (hGSTO1)30.
In order to determine the structure of a complex between TvGSTO6S and a avonoid, several candidate mol-
ecules were chosen for soaking experiments. Crystals of TvGSTO6S were unstable and brittle, and required some
tricks to prepare complexes. Among the trials performed, one was successful where the droplet that gave rise
to TvGSTO6S crystals was deposited for a few hours on a surface previously coated with naringenin (chemical
structure in Supplementary TableS4). e structure of the complex was solved at a resolution of 2.3 Å (Figs1 and
4, Supplementary TableS1). Two naringenin molecules bind in the pocket described above, which constitutes the
L-site of TvGSTO6S (Figs1 and 4). Each naringenin aromatic ring stacks with its symmetrical equivalent in an
energetically-favored association (−13.64 kcal/mol calculated by DFT). A pair of stacked avonoids was already
observed bound to dihydroavonol 4-reductase, however in a head to tail arrangement in the active site of the
Figure 1. Overall views of Trametes versicolor GSTO3S structure in complex with glutathione and 2,4 hydroxy
benzophenone (le panels) and GSTO6S structure in complex with naringenin (right panels). In each case,
structures are depicted in cartoon mode with ligands shown as spheres and sticks (glutathione in green, 2,4-
HBP and naringenin in yellow). N-terminal domains are shown in light colors (white for GSTO3S, cyan for
GSTO6S) and C-terminal domains are shown in deeper colors (grey for GSTO3S, blue for GSTO6S). Black
arrows indicate positions of glutathione binding site (G-site), hydrophobic binding site (H-site) and ligandin
site (L-site). In each case is represented one physiological dimer that typies the structure of GSTs where the
N-terminal domain (secondary structure β1α1β2α2β3β4α3) of one monomer cross-interacts with the C-terminal
domain (α4α5α6α6′α7α8α9) of the second one, and vice-versa.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
5
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
enzyme, with no dimer symmetry31. In TvGSTO6S, the naringenin pair ts the apolar environment formed by
the neighboring aliphatic part of the side chains of both monomers (L84, V85, R111, T115, E118 and T119) while
a hydrogen bond with E88 stabilizes one of the avonoid hydroxyl groups. L-sites at the dimer interface were
previously described for hGSTO1 (pdb code 4is0)30 and for Arabidopsis thaliana GST Phi 2 (AtGSTF2, pdb code
5a4v)32 however with one ligand only. In hGSTO1, the L-site takes place at a location similar to TvGSTO6S and
shows conserved patches of hydrophobic residues along helix α3 (Supplementary Fig.S2). Interestingly, E88 that
stabilizes naringenin in TvGSTO6S is conserved in hGSTO1 (E91) where its side chain is found near the nitro-
phenacyl moiety of the glutathione adduct. e case of AtGSTF2 is quite dierent. A quercetin molecule sits at
the opposite side of the dimer interface, near the C-terminal end of helix α3 and the N-terminal end of helix α4.
Structural comparisons suggest that Omega GSTs have two hydrophobic ligand binding sites: an H-site near
α4 and α6 and an L-site at the dimer interface. In the case of TvGSTO6S, the L-site can host avonoids. e
alignment of the solved structures with the sequences of TvGSTO1S, −2S, −4S and −5S shows variations at the
putative H- and L-sites (Supplementary Fig.S2), which could explain the isoform-specic TSA patterns.
Figure 2. Binding of 2,4- and 2,4,4′-hydroxy benzophenones in GSTO3S hydrophobic binding site. Stereoviews
of sections of the GSTO3S complex structures with 2,4-HBP (top view) and 2,4,4′-HBP (bottom view) are
shown. TvGSTO3S H-site is a well-delineated cavity deeply inserted in between helices α4 and α6 of the
C-terminal domain. It is perfectly suited to accommodate polyaromatic ligands, due to its strong hydrophobic
character given by the aromatic side chains of F123, W127, F128, F168, the aliphatic parts of R171 and Y175,
completed with polar residues (Y17 and R124) at the cavity entrance close to the G-site. Polar intermolecular
contacts are materialized as dashed lines. Surrounding side chains are represented in sticks. HBPs are shown
as yellow sticks and spheres. 2mFo-DFc composite omit maps shown at 1.0 σ around HBPs were calculated by
PHENIX.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
6
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
Fishing of a avonoid from a wild-cherry heartwood extract by anity crystallography. One of
the biggest challenges in GST characterization is the identication of natural ligands33. Anity crystallography is
a very new method that was recently conceived to select and identify new inhibitors from natural crude extracts
as potent drug scaolds for pharmaceutical targets34. We successfully applied this method on TvGSTO3S crystals
to isolate the avonoid dihydrowogonin from a partially puried wild-cherry extract.
Acetonic extracts of wild-cherry heartwood were fractioned by reverse chromatography. e potential inhi-
bition of the various fractions on TvGSTO esterase activity was analyzed using both chloromethyluorescein
diacetate(CMFDA) and methylumbelliferyl acetate (MUA)as substrates (Fig.5). In previous studies13,19, these
substrates had increased detection sensitivity of uorescence and avoided quenching eects of wood extracts. e
fractions that eluted aer 46 and 47 min induced a strong inhibition of both measured activities. e pooled mix-
ture was tested for its ability to interact with TvGSTO3S using TSA. A positive 4.8 °C shi of the observed ∆Td
conrmed the presence of potential TvGSTO3S ligands. Analysis of this solution by LC-MS revealed the presence
of at least two compounds with molecular masses of 254 and 286 g.mol−1 respectively (Fig.5). e 46–47 min
eluate also analysed by H1NMR (spectrum shown in Fig.5) exhibited characteristic signals of a avanone skeleton
with, in addition, typical methyloxy group singlets at 3.75 or 3.81 ppm. At this stage, several compounds already
described in the extractives composition could correspond to such data (e.g. dihydrowogoninor sakuranetin)35–37
so that it was impossible to denitively assign the molecular structures, particularly concerning the MeO group
position either on the ring A or B (Fig.5).
Anity crystallography was then used to further elucidate the interactions between TvGSTO3S and com-
pounds present in the 46–47 min eluate. is approach assumes that a complex can form when the protein is
mixed with a partially puried mixture of molecules containing potential ligands. We re-suspended the dried
eluate with a minimal volume of DMSO to obtain a concentrated mixture suitable for TvGTO3S crystalliza-
tion. e addition of 2.5% of the concentrated solution in the mother liquor produced co-crystals as observed
from the electron density peak in the H-site. High quality of the signal together with the NMR and MS data led
to unambiguous identication of dihydrowogonin (Fig.5). is avonoid was previously identied in Prunus
avium heartwood and described as a avanone35. Interestingly, its corresponding avone (wogonin) was shown to
strongly inhibit the GSH-transferase activity of TvGSTO3S towards PEITC (KI = 2.89 ± 1.24 µM, Supplementary
Figure 3. Structural comparison of TvGSTO3S and −6S isoforms showing dierences between H-sites and
L-sites. e structures shown are TvGSTO3S in complex with 2,4-HBP (top le) and TvGSTO6S in complex
with naringenin (top right). In both cases, helix α9 and C-terminal tail were removed from structures for clarity.
Monomers A and B are colored white and grey, respectively. 2,4-HBP and naringenin molecules are represented
as yellow sticks. H-site and L-sites residues are colored respectively cyan and grey in the structures (highlighted
respectively cyan and grey on the structural alignment). G-site residues are highlighted green in the sequences
and are not shown on structures for clarity. Catalytic serine is highlighted yellow in the sequences. Secondary
structures are labelled and shown using arrows (β-strands) and squiggles (helices). Secondary structure
elements are based on TvGSTO6S. Helix α9 extra turns in TvGSTO6S are colored red on the sequence
alignment.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
7
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
TableS5), while no inhibition was detected for TvGSTO6S. Since in the latter a stabilizing eect is still observed
by TSA, the ligand should not bind in its catalytic site (i.e. H-site). In TvGSTO3S, the unsubstituted ring of
dihydrowogonin sits in the bottom of the H-site like the ring B of HBPs (see above) (Supplementary Fig.S7).
e opposite ring points towards the entrance of the cavity the polar residues of which stabilize its methoxy and
hydroxyl groups by direct or water-mediated hydrogen bonds. e hydroxyl group situated at position 7 is only
2.6 Å from the glutathione sulfur atom. is short distance questions a possible catalysis with a related substrate.
e combined use of X-ray diraction, NMR and MS succeeded in identifying dihydrowogonin as a natural mol-
ecule that originates from wild-cherry heartwood and that tightly binds TvGSTO3S.
Conclusion
In this study, we demonstrated at the biochemical and structural level that T. versicolor GSTs that belong to the
Omega class interact with polyphenolic compounds found in wood, and in particular with avonoids such as
dihydrowogonin and naringenin. Indeed, white-rot fungi such as T. versicolor have to cope with potentially toxic
tree secondary-metabolites mainly constituted of polyphenolic compounds, which accumulate in dierent parts
of the wood (heartwood, knots). e molecular interactions between TvGSTOs and polyphenols appear to be
very diverse. ey potentially involve two structural sites for each isoform. It is tempting to establish a correlation
between the diversity of these interactions and the extension of the Omega class (at least 16 isoforms) in T. versi-
color, which encounters a large diversity of polyphenols in its natural environment. However, the exact function
of this GST network remains unclear since no catalytic activity against the tested polyphenols has been detected.
e ability of TvGSTOs to bind polyphenols through dierent sites suggests that fungal GSTs could be involved
in the transport of various polyphenols. In plants, GSTs were shown to facilitate the avonoid transport from the
cytoplasm into the vacuole38. ese GSTs could act as avonoid and glutathione carriers, providing ABC trans-
porters with both molecules for their co-transport. is transport requires free glutathione but no glutathionyla-
tion activity39. Our study suggests that fungal GSTs as their plant homologues could be involved in the transport
and sequestration of avonoids.
Methods
Reagents. All pure molecules together with the uorescent marker SYPRO® Orange used in TSA were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA), except for wogonin provided by Extrasynthese (Genay, France).
Production and purication of proteins. e production in E. coli (Rosetta2 DE3 pLysS strain, Novagen)
and purication of the six selected TvGSTOs (accession number in the JGI database: TvGSTO1S: Tv75639;
TvGSTO2S: Tv56280; TvGSTO3S: Tv48691; TvGSTO4S: Tv65402; TvGSTO5S: Tv54358 and TvGSTO6S:
Tv23671) were performed as explained previously19.
Study of the thermostability of TvGSTOs. e experiments were performed in 96 well microplates
(Harshell, Biorad) and the measurements carried out using a real time PCR detection system (CFX 96 touch,
Biorad). e assays were performed as follows: 5 μL of Tris-HCl (150 mM) pH 8 buer, 2 μL of pure molecules
dissolved in DMSO (nal concentration: 0.8 mg/mL for molecules of the chemical library; 100 µM for benzo-
phenones and avonoids), 5 μL of proteins (contained in Tris-HCl 150 mM, pH 8; nal concentration: 40 µM for
Figure 4. Binding of naringenin in GSTO6S non-catalytic ligandin site. Stereoview of a section of the GSTO6S
complex structure with a pair of naringenin molecules is shown. GSTO6S ligandin site is an hydrophobic pocket
inserted at the interface of the dimer, between helix α3 and α4 of both monomers around the dimer 2-fold axis.
e apolar environment is formed by the aliphatic part of the side chains of both monomers (L84, V85, T115,
E118) while a hydrogen bond with E88 stabilizes one of the avonoid hydroxyl groups. Polar intermolecular
contacts are materialized as dashed lines. Surrounding side chains are represented in sticks. Naringenin is
shown as yellow sticks and spheres. 2mFo-DFc composite omit map shown at 1.0 σ around the ligand was
calculated by PHENIX.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
8
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
the tests with the chemical library; 10 μM for the other tests), 2 μL of SYPRO orange (previously diluted 80−fold
in ultra-pure water) and 11 μL of ultra-pure water for a total volume of 25 μL per well. e plate was centrifuged
30 seconds at 4000 g. Fluorescence was measured (excitation: 485 nm; emission: 530 nm) every minute starting
from 3 minutes at 5 °C while increasing temperature from 5 to 95 °C with a step of 1 °C per minute. e denatur-
ation temperature (Td), which corresponds to the temperature where 50% of the total uorescence is measured,
was determined by using the non-linear regression Boltzmann sigmoidal model in GraphPad Prism 6 soware
for data obtained in presence of potential ligands, while the reference was considered for similar experiments
conducted by adding DMSO only.
Inhibition Kinetics. Competition tests between phenethyl isothiocyanate (PEITC) and benzophenones or
avonoids were performed in a nal volume of 500 µL. Inhibition tests of the glutathionylation activity were
assayed with various concentrations of PEITC (25–300 µM) and a xed concentration of GSH (1 mM) in presence
or not of inhibitors. ese experiments were carried out in 100 mM pH 6.4 phosphate buer at 25 °C by analyzing
the glutathionylated product which absorbs at 274 nm. Basal activity of samples containing GSH, PEITC and ben-
zophenones was subtracted from the enzyme catalyzed rates. e inhibition constants (Ki) were calculated using
the GraphPad soware with the nonlinear regression based on the mixed model inhibition.
Inhibition tests of the esterase activity of TvGSTO3S with CMFDA or MUA as substrates in presence of extract
fractions were performed in 96-well microplates. e emission of uorescence at 517 nm and 460 nm aer an excita-
tion at 492 nm and at 355 nm for CMFDA and 4-MUA respectively was followed using microplate reader (2030
Multilabel Reader Victor X5, PerkinElmer). Reactions were performed in 200 µL of Tris-HCl pH 8 (30 mM), EDTA
(1 mM) buer, with 0.5 µM of CMFDA, 5 µM of 4-MUA and 1 mM of GSH. Using both uorescent substrates,
TvGSTO3S activity was measured in presence or in absence of 2 µL of the tested fractions. e ratio between the two
slopes (∆RFU/min, Relative Fluorescence Unit) has been used to determine the potential inhibition.
Figure 5. Combined approach including anity crystallography revealed dihydrowogonin bound to GSTO3S
hydrophobic site. (A) Normalized inhibition of esterase activity with substrates CMFDA and MUA is shown.
Fractions 46–47 min that inhibited both activities were selected for further analysis. (B) MS analysis in positive
(bottom panel) and negative (top panel) modes revealed two major compounds. (C) 1H-NMR spectrum
showed the structural features of avanones. Anity crystallography allowed the elucidation of the avanone
dihydrowogonin. Its 1H-NMR data37 are indeed found on the spectrum of the mixture: numbers in parenthesis
are the typical chemical shis for dihydrowogonin and numbers in blue correspond to the values obtained
in the present study. Integration values of 2 methoxy groups allowed to evaluate maximum abundance of
dihydrowogonin in the fractions (numbers in red). (D) Electron density of dihydrowogonin in structure of
GSTO3S crystallized in presence of 46-47 eluate. e map shown is a 2mFo-DFc composite omit map contoured
at 1 σ. (E) Chemical structure of dihydrowogonin.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
9
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
Fractionation of wild-cherry hardwood acetonic extracts by HPLC. Wild-cherry hardwood acetonic
extracts were fractioned by high-performance liquid chromatography. 100 µL of extract at 10 mg/mL were fractioned
by reverse chromatography using Kinetex biphenyl column (250 × 4,6 mm) previously equilibrated with H2O/
Formic acid 0.1% buer. e molecules adsorbed to the column were eluted with the help of a gradient of methanol
(from 0 to 100%). Collected fractions were evaporated using SpeedVac™ (UniEquip) and nally dissolved in DMSO.
Crystallogenesis experiments. Crystallization of TvGSTO3S was assayed by the microbatch under oil
method at 278 K. TvGSTO3S (13 mg/mL) crystallized by mixing 1 µL of protein with 3 µL of commercial solu-
tion consisting in 30% (w/v) PEG 400, 0.2 M calcium acetate in 0.1 M pH 4.5 acetate buer (Wizard Classic
Screen 1, Rigaku). Crystals of the complexes of TvGSTO3S with glutathione (TvGSTO3S - GSH), TvGSTO3S
with glutathionyl-dinitrobenzene (TvGSTO3S - GS-DNB), TvGSTO3S with hexyl-glutathione (TvGSTO3S
- GS-hexyl) and TvGSTO3S with glutathionyl-phenylacetophenone (TvGSTO3S - GS-PAP) were obtained
by soaking apo TvGSTO3S crystals during one hour in the mother liquor containing 10 mM ligand (0.5 mM
in the case of GS-PAP). Crystals of the complexes of TvGSTO3S with HBPs (2,3,4-trihydroxybenzophenone,
3,4-dihydroxybenzophenone, 2,4-dihydroxybenzophenone and 2,4,4′-trihydroxybenzophenone) were obtained
by co-crystallizing the protein pre-incubated for 30 min with 10 mM ligand. Complex of TvGSTO3S with dihydro-
wogonin was obtained in a similar way by using 10 mg/mL of partially-puried wild-cherry hardwood extract.
First screening of the TvGSTO6S crystallization conditions was performed by using an Oryx 8 robot (Douglas
Instruments) to implement sitting drops with commercial kits of various solutions. Crystals were then optimized
by the hanging drop method. e droplet was prepared by mixing 1 µL of TvGSTO6S (26 mg/mL) with 0.2 µL of
a crystal seed stock obtained by crushing the droplet content of the best hits of the screening step and with 1 µL
of a crystallization solution consisting in 25% (w/v) PEG 1500, 0.1 M pH 6.5 MMT buer (containing DL-malic
acid, MES and Tris base in the molar ratios 1:2:2, respectively). e reservoir contained 1 mL of the same crys-
tallization condition. Attempts using classical co-crystallization or soaking experiments failed to prepare crys-
tals of TvGSTO6S complexes with avonoids. A new strategy was developed based on the dry co-crystallization
method40. In this original ‘dry soaking’ technique, 0.2 µL of naringenin (100 mM) solubilized in DMSO was
deposited on a cover slide and le to complete evaporation. en, one TvGSTO6S crystal together with 1 µL of its
mother liquor was dispensed on the dried naringenin allowing partial ligand resolubilization. e cover slide was
then reinstalled above the reservoir that initially allowed crystallization until crystal harvest (ca. 1 day).
Data collection, processing and renement. TvGSTO3S and −6S crystals were ash-frozen aer a quick
soaking in their mother liquor complemented with 20% (v/v) glycerol as cryoprotectant. Primary X-ray diraction
experiments were carried out in-house on a laboratory diractometer (Agilent SuperNova with CCD detector).
Data collection up to 2.5 Å resolution allowed preliminary analysis, especially for ligand screening in TvGSTO3S
active site. High resolution diraction experiments were carried out on the ESRF beamlines FIP BM30A and
ID30B (Grenoble, France). TvGSTO3S and −6S native crystals diracted up to 1.35 Å and 1.48 Å, respectively. Data
sets were indexed, integrated and scaled with XDS41. e structure of TvGSTO3S was solved by molecular replace-
ment using MR BUMP automated pipeline from CCP4 suite42 with the coordinates of poplar GST Lambda 3 (PDB
code 4PQI) as the search model. e electron density of a small molecule that we failed to identify was observed in
the H-site. It probably bound to the enzyme during the purication process and was not modelled in the electron
density. Some of the structures of TvGSTO3S in complex with ligands displayed electron density corresponding to
glutathione remaining from the purication process. When present, this residual GSH was modelled, sometimes
with partial occupancy. e structure of TvGSTO6S was solved by molecular replacement using PHASER with
the coordinates of TvGSTO3S. All structures were rened with PHENIX43 and built with COOT44. Restraint les
for ligands were generated with phenix.elbow and the grade server (URL http://grade.globalphasing.org/cgi-bin/
grade/server.cgi). In all of the concerned structures, the occupancies of the ligands added by co-crystallization
or soaking techniques were set to 1 and the corresponding B factors were compatible with full presence of the
molecules in their binding sites (except for 2,3,4-HPB where the occupancy is 0.8). Validation of all structures was
performed with MolProbity45 and the wwPDB validation service (http://validate.wwpdb.org). Coordinates and
structure factors have been deposited in the Protein Data Bank. Data-collection and renement statistics of all
structures are shown in Supplementary TableS1. Stereo images of a portion of 2mFo-DFc electron density maps are
shown in Supplementary TableS6 to assess quality of the structural data. All gures were prepared by using Pymol
(e PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Density functional theory (DFT) calculation of naringenin dimer stabilization in TvGSTO6S
(Gaussian09 software). e molecular structure of the dimer of naringenin molecules was extracted from
the experimental X-ray structure of TvGSTO6S. Geometry optimization of the hydrogen atoms alone was then
performed46 at the DFT level of theory in vacuum (i.e. no environment eect were taken into account), employing
the B3LYP functional47 completed with Grimme’s D3 dispersion correction48; the 6–111 G(d,p) basis set was used,
and basis set superposition errors were corrected by the counterpoise method of Bernardi49. e complexation
energy at the converged geometrical structure was −13.64 kcal/mol, indicating a strong stabilization of the narin-
genin molecules within their dimer as found in TvGSTO6S.
Data Availability. e atomic coordinates of the crystal structures from this publication have been deposited
to the Protein Data Bank (https://www.rcsb.org) and assigned the PDB codes 6F43, 6F4B, 6F4F, 6F4K, 6F51, 6F66,
6F67, 6F68, 6F69, 6F6A, 6F70 and 6F71.
Accession codes. Accession numbers of TvGSTOs in the JGI database are as follows: TvGSTO1S: Tv75639;
TvGSTO2S: Tv56280; TvGSTO3S: Tv48691; TvGSTO4S: Tv65402; TvGSTO5S: Tv54358 and TvGSTO6S: Tv23671.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
10
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
References
1. Tien, M. & ir, T. . Lignin-Degrading Enzyme From e Hymenomycete Phanerochaete-Chrysosporium Burds. Science 221,
661–662, https://doi.org/10.1126/science.221.4611.661 (1983).
2. Floudas, D. et al. e Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336,
1715–1719, https://doi.org/10.1126/science.1221748 (2012).
3. Eastwood, D. C. et al. e Plant Cell Wall-Decomposing Machinery Underlies the Functional Diversity of Forest Fungi. Science 333,
762–765, https://doi.org/10.1126/science.1205411 (2011).
4. Nagy, L. G. et al. Genetic Bases of Fungal White ot Wood Decay Predicted by Phylogenomic Analysis of Correlated Gene-
Phenotype Evolution. Molecular biology and evolution 34, 35–44, https://doi.org/10.1093/molbev/msw238 (2017).
5. Harms, H., Schlosser, D. & Wic, L. Y. Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nature
Reviews Microbiology 9, 177–192, https://doi.org/10.1038/nrmicro2519 (2011).
6. Morel, M. et al. Xenomic networs variability and adaptation traits in wood decaying fungi. Microbial biotechnology 6, 248–263,
https://doi.org/10.1111/1751-7915.12015 (2013).
7. Valette, N., Perrot, T., Sormani, ., Gelhaye, E. & Morel-ouhier, M. Antifungal activities of wood extractives. Fungal Biology
Reviews 31, 113–123, https://doi.org/10.1016/j.r.2017.01.002 (2017).
8. Syed, ., Shale, ., Pagadala, N. S. & Tuszynsi, J. Systematic Identication and Evolutionary Analysis of Catalytically Versatile
Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi. Plos One 9, https://doi.org/10.1371/journal.
pone.0086683 (2014).
9. Enayati, A. A., anson, H. & Hemingway, J. Insect glutathione transferases and insecticide resistance. Insect molecular biology 14,
3–8, https://doi.org/10.1111/j.1365-2583.2004.00529.x (2005).
10. Schuler, M. A. & Berenbaum, M. . Structure and Function of Cytochrome P450S in Insect Adaptation to Natural and Synthetic
Toxins: Insights Gained from Molecular Modeling. Journal of Chemical Ecology 39, 1232–1245, https://doi.org/10.1007/s10886-013-
0335-7 (2013).
11. ane, . V. et al. Are feeding preferences and insecticide resistance associated with the size of detoxifying enzyme families in insect
herbivores? Current Opinion in Insect Science 13, 70–76, https://doi.org/10.1016/j.cois.2015.12.001 (2016).
12. Morel, M., Ngadin, A. A., Droux, M., Jacquot, J. P. & Gelhaye, E. e fungal glutathione S-transferase system. Evidence of new classes
in the wood-degrading basidiomycete Phanerochaete chrysosporium. Cellular and molecular life sciences: CMLS 66, 3711–3725,
https://doi.org/10.1007/s00018-009-0104-5 (2009).
13. Mathieu, Y. et al. Diversication of Fungal Specic Class A Glutathione Transferases in Saprotrophic Fungi. Plos One 8, https://doi.
org/10.1371/journal.pone.0080298 (2013).
14. Marinović, M. et al. Selective Cleavage of Lignin β-O-4 Aryl Ether Bond by β-Etherase of the White-ot Fungus Dichomitus
squalens. ACS Sustainable Chemistry & Engineering 6, 2878–2882, https://doi.org/10.1021/acssuschemeng.7b03619 (2018).
15. oret, T. et al. Evolutionary divergence of Ure2pA glutathione transferases in wood degrading fungi. Fungal genetics and biology: FG
& B 83, 103–112, https://doi.org/10.1016/j.fgb.2015.09.002 (2015).
16. ontur, W. S. et al. Novosphingobium aromaticivorans uses a Nu-class glutathione-S-transferase as a glutathione lyase in breaing
the beta-aryl ether bond of lignin. e Journal of biological chemistry, https://doi.org/10.1074/jbc.A117.001268 (2018).
17. Board, P. G. et al. Identication, characterization, and crystal structure of the Omega class glutathione transferases. e Journal of
biological chemistry 275, 24798–24806, https://doi.org/10.1074/jbc.M001706200 (2000).
18. Meux, E. et al. New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases. Biochimie 95, 336–346,
https://doi.org/10.1016/j.biochi.2012.10.003 (2013).
19. Deroy, A. et al. The GSTome eflects the Chemical Environment of White-ot Fungi. Plos One 10, e0137083, https://doi.
org/10.1371/journal.pone.0137083 (2015).
20. Lo, M. C. et al. Evaluation of uorescence-based thermal shi assays for hit identication in drug discovery. Anal Biochem 332,
153–159, https://doi.org/10.1016/j.ab.2004.04.031 (2004).
21. Wu, S. B., Long, C. L. & ennell, E. J. Structural diversity and bioactivities of natural benzophenones. Natural product reports 31,
1158–1174, https://doi.org/10.1039/c4np00027g (2014).
22. Mosedale, J. . & Puech, J. L. Wood maturation of distilled beverages. Trends in Food Science & Technology 9, 95–101, https://doi.
org/10.1016/s0924-2244(98)00024-7 (1998).
23. rissinel, E. & Henric, . Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.
Acta Crystallographica Section D-Biological Crystallography 60, 2256–2268, https://doi.org/10.1107/s0907444904026460 (2004).
24. om, . et al. Structure of a tau class glutathione S-transferase from wheat active in herbicide detoxication. Biochemistry 41,
7008–7020, https://doi.org/10.1021/bi015964x (2002).
25. Yamamoto, ., Suzui, M., Higashiura, A. & Naagawa, A. ree-dimensional structure of a Bombyx mori Omega-class glutathione
transferase. Biochem Biophys Res Commun 438, 588–593, https://doi.org/10.1016/j.bbrc.2013.08.011 (2013).
26. Oaley, A. Glutathione transferases: a structural perspective. Drug Metab Rev 43, 138–151, https://doi.org/10.3109/03602532.2011
.558093 (2011).
27. C ox, P. J., echagias, D. & elly, O. Conformations of substituted benzophenones. Acta crystallographica. Section B, Structural science
64, 206–216, https://doi.org/10.1107/S0108768108000232 (2008).
28. Badia-Fabregat, M. et al. Degradation of UV lters in sewage sludge and 4-MBC in liquid medium by the ligninolytic fungus
Trametes versicolor. Journal of environmental management 104, 114–120, https://doi.org/10.1016/j.jenvman.2012.03.039 (2012).
29. Janjusevic, L. et al. e lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE
inhibitory agents. Journal of enzyme inhibition and medicinal chemistry 32, 355–362, https://doi.org/10.1080/14756366.2016.12527
59 (2017).
30. Broc, J., G., B. P. & Oaley, A. J. Structural insights into omega-class glutathione transferases: a snapshot of enzyme reduction and
identication of a non-catalytic ligandin site. Plos One 8, https://doi.org/10.1371/journal.pone.0060324.g001 (2013).
31. Trabelsi, N. et al. Structural evidence for the inhibition of grape dihydroavonol 4-reductase by avonols. Acta crystallographica.
Section D, Biological crystallography D64, 883–891, https://doi.org/10.1107/S0907444908017769 (2008).
32. Ahmad, L., ylott, E. L., Bruce, N. C., Edwards, . & Grogan, G. Structural evidence for Arabidopsis glutathione transferase
AtGSTF2 functioning as a transporter of small organic ligands. Febs Open Bio 7, 122–132, https://doi.org/10.1002/2211-5463.12168
(2017).
33. Mashiyama, S. T. et al. Large-scale determination of sequence, structure, and function relationships in cytosolic glutathione
transferases across the biosphere. PLoS biology 12, e1001843, https://doi.org/10.1371/journal.pbio.1001843 (2014).
34. Aguda, A. H. et al . Anity Crystallography: A New Approach to Extracting High-Anity Enzyme Inhibitors from Natural Extracts.
Journal of natural products 79, 1962–1970, https://doi.org/10.1021/acs.jnatprod.6b00215 (2016).
35. McNult y, J. et al. Isolation of flavonoids from the heartwood and resin of Prunus avium and some preliminary biological
investigations. Phytochemistry 70, 2040–2046, https://doi.org/10.1016/j.phytochem.2009.08.018 (2009).
36. ebbi-Beneder, Z., Colin, F., Dumarcay, S. & Gerardin, P. Quantication and characterization of notwood extractives of 12
European sowood and hardwood species. Annals of Forest Science 72, 277–284, https://doi.org/10.1007/s13595-014-0428-7 (2015).
37. Vinciguerra, V., Luna, M., Bistoni, A. & Zollo, F. Variation in the composition of the heartwood avonoids of Prunus avium by on-
column capillary gas chromatography. Phytochemical Analysis 14, 371–377, https://doi.org/10.1002/pca.730 (2003).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
11
Scientific REPoRtS | (2018) 8:8472 | DOI:10.1038/s41598-018-26601-3
38. Dixon, D. P. & Edwards, . oles for stress-inducible lambda glutathione transferases in avonoid metabolism in plants as identied
by ligand shing. e Journal of biological chemistry 285, 36322–36329, https://doi.org/10.1074/jbc.M110.164806 (2010).
39. Zhao, J. Flavonoid transport mechanisms: how to go, and with whom. Trends in Plant Science 20, 576–585, https://doi.org/10.1016/j.
tplants.2015.06.007 (2015).
40. Gelin, M. et al. Combining ‘dry’ co-crystallization and in situ diraction to facilitate ligand screening by X-ray crystallography. Acta
crystallographica. Section D, Biological crystallography 71, 1777–1787, https://doi.org/10.1107/S1399004715010342 (2015).
41. absch, W. X. Acta crystallographica. Section D, Biological crystallography 66, 125–132, https://doi.org/10.1107/S0907444909047337
(2010).
42. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta crystallographica. Section D, Biological crystallography
67, 235–242, https://doi.org/10.1107/S0907444910045749 (2011).
43. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica.
Section D, Biological crystallography 66, 213–221, https://doi.org/10.1107/S0907444909052925 (2010).
44. Emsley, P. & Cowtan, . Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological
crystallography 60, 2126–2132, https://doi.org/10.1107/S0907444904019158 (2004).
45. Davis, I. W., Mur ray, L. W., ichardson, J. S. & ichardson, D. C. MOLPOBITY: structure validation and all-atom contact analysis
for nucleic acids and their complexes. Nucleic Acids Res 32, W615–619, https://doi.org/10.1093/nar/gh398 (2004).
46. Frisch, M. J. et al. (Wallingford CT, 2009).
47. Bece, A. D. Densityfunctional thermochemistry. III. e role of exact exchange. e Journal of Chemical Physics 98, 5648–5652,
https://doi.org/10.1063/1.464913 (1993).
48. Grimme, S., Antony, J., Ehrlich, S. & rieg, H. A consistent and accurate ab initio parametrization of density functional dispersion
correction (DFT-D) for the 94 elements H-Pu. e Journal of Chemical Physics 132, 154104, https://doi.org/10.1063/1.3382344
(2010).
49. B oys, S. F. & Bernardi, F. e calculation of small molecular interactions by the dierences of separate total energies. Some procedures
with reduced errors. Molecular Physics 19, 553–566, https://doi.org/10.1080/00268977000101561 (1970).
Acknowledgements
We thank Solène Telliez, Tiphaine Dhalleine, Jean-Michel Girardet and Sandrine Mathiot for technical assistance.
A sincere thank you to Pr. Jean-Pierre Jacquot for constructive criticism of the manuscript. is work was granted
access to the HPC resources of CCRT/CINES/IDRIS under the allocation A0030807449 made by GENCI. e
authors would like to thank ESRF for beamtime, and the sta of beamlines BM30A and ID30B for assistance with
crystal testing and data collection. e authors appreciated the access to the ‘Plateforme de mesures de diraction
X’ of the Université de Lorraine. is study was funded by the French National Research Agency (ANR-11-
LAS-0002-01), the Centre National de la Recherche Scientique, the University of Lorraine and the Région Grand
Est (MS and TP Grants, PEPS-Mirabelle 2016, CPER 2014-2020, Program “Equipement mi-lourd 2016”).
Author Contributions
F.F., E.G. and C.D. developed the concept and supervised this study. E.A., T.P., M.S., S.D., M.M.R., F.S., G.M.
performed the experiments and interpreted the data. All the authors participated in manuscript writing. E.G.,
C.D. and P.G. acquired the funding. All authors read and approved the nal manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26601-3.
Competing Interests: e authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© e Author(s) 2018
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com