Glutathione transferases of Phanerochaete chrysosporium: S-glutathionyl-p-hydroquinone reductase belongs to a new structural class

Article abstract of DOI:10.1074/jbc.M110.194548

The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.

Full text of DOI:10.1074/jbc.M110.194548

Protein Structure and Folding:
Glutathione Transferases of Phanerochaete
chrysosporium :
S-GLUTATHIONYL-p-HYDROQUINONE
REDUCTASE BELONGS TO A NEW
STRUCTURAL CLASS
Edgar Meux, Pascalita Prosper, Andrew
Ngadin, Claude Didierjean, Mélanie Morel,
Stéphane Dumarçay, Tiphaine Lamant,
Jean-Pierre Jacquot, Frédérique Favier and
Eric Gelhaye
J. Biol. Chem. 2011, 286:9162-9173.
doi: 10.1074/jbc.M110.194548 originally published online December 22, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 11, pp. 9162–9173, March 18, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Glutathione Transferases of Phanerochaete chrysosporium
S-GLUTATHIONYL-p-HYDROQUINONE REDUCTASE BELONGS TO A NEW STRUCTURAL
□
S
CLASS*
Received for publication, October 14, 2010, and in revised form, December 17, 2010 Published, JBC Papers in Press, December 22, 2010, DOI 10.1074/jbc.M110.194548
Edgar Meux‡, Pascalita Prosper§, Andrew Ngadin‡, Claude Didierjean§, Me´lanie Morel‡, Ste´phane Dumarc¸ay¶,
Tiphaine Lamant‡, Jean-Pierre Jacquot‡, Fre´de´rique Favier§1, and Eric Gelhaye‡2
From the ‡UMR 1136 INRA-UHP “Interactions Arbres/Micro-Organismes,” IFR110 “Ecosyste`mes Forestiers, Agroressources,
Bioproce´de´s et Alimentation,” the §CRM2, Equipe Biocristallographie, UMR 7036 CNRS-UHP, Institut Jean Barriol, and the
¶
´
Laboratoire d’Etudes et de Recherches sur le Mate´riau Bois, EA UHP 4370, Nancy Universite´, Faculte´ des Sciences et Techniques,
BP 70239, 54506 Vandoeuvre-les-Nancy, France
The white rot fungus Phanerochaete chrysosporium, a sapro-
phytic basidiomycete, possesses a large number of cytosolic glu-
tathione transferases, eight of them showing similarity to the
Omega class. PcGSTO1 (subclass I, the bacterial homologs of
which were recently proposed, based on their enzymatic func-
tion, to constitute a new class of glutathione transferase named
S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3
(subclass II related to mammalian homologs) have been investi-
gated in this study. Biochemical investigations demonstrate that
both enzymes are able to catalyze deglutathionylation reactions
thanks to the presence of a catalytic cysteinyl residue. This reac-
tion leads to the formation of a disulfide bridge between the
conserved cysteine and the removed glutathione from their
substrate. The substrate specificity of each isoform differs. In
particular PcGSTO1, in contrast to PcGSTO3, was found to
catalyze deglutathionylation of S-glutathionyl-p-hydroquinone
substrates. The three-dimensional structure of PcGSTO1 pre-
sented here confirms the hypothesis that it belongs not only to a
new biological class but also to a new structural class that we
propose to name GST xi. Indeed, it shows specific features, the
most striking ones being a new dimerization mode and a cata-
lytic site that is buried due to the presence of long loops and that
contains the catalytic cysteine.
to carbon dioxide and water (1–3). Analysis of the genome of
this basidiomycete revealed a complex superfamily of cyto-
chrome P450 monooxygenases representing around 1% of this
genome (1, 4). The large size of this gene family is correlated
with the extraordinary ability of this fungus to degrade and
metabolize numerous recalcitrant compounds such as pesti-
cides, polyaromatic hydrocarbons, halogenated aromatics, and
textile dyes. Besides extracellular oxidative systems, including
lignin and manganese peroxidases, cytochrome P450 monooxyge-
nases are thought to be involved in the phase I of detoxication
processes catalyzing the oxidation of various substrates (5–7). The
oxidation of recalcitrant compounds is often followed by glutathi-
one conjugation catalyzed by glutathione transferases (GSTs). The
glutathione conjugates formed are less toxic and lipophilic than
the parent compounds (8).
The distribution of GSTs in fungi has been recently investi-
gated (9), highlighting that P. chrysosporium exhibits a large
subfamily of cytosolic GSTs (at least 27 isoforms) in compari-
son with Saccharomyces cerevisiae for instance (11 isoforms).
Based on primary structure similarity, these cytosolic GSTs
belong to seven different classes as follows: Ure2p-like, GTT1,
GTT2, EFB␥, MAK16, etherases, and Omega (GSTO)3 (9).
Among the cytosolic GSTs, P. chrysosporium exhibits eight
putative proteins showing homology with GSTs of the Omega
class, named PcGSTO1 to PcGSTO8. In a previous phyloge-
Phanerochaete chrysosporium is considered as the model netic analysis of fungal GSTs, we found that GSTOs fall into
organism to study the physiology of white rot fungi, the only three different subclasses. Subclass 1 encloses PcGSTO1 and
known microorganisms able to completely break down lignin PcGSTO2, these latter being related to uncharacterized plant
GST Lambda and bacterial GSTs (9). Members of subclasses II
* This work was supported by Agence Nationale de la Recherche Research
Grants ANR-06-BLAN-0386 and ANR-09-BLAN-0012, Ministe`re de
l’Enseignement Supe´rieur de la Recherche et de la Technologie, Institut
and III are related to the well characterized human HsGSTO1-1
(10, 11).
A major feature of GSTOs is the presence of a cysteinyl res-
National de la Recherche Agronomique, and CNRS.
idue in the catalytic site present in the glutathione binding
This work is dedicated to the memory of the late Dr. Andre´ Aubry.
□S
domain (G-site), explaining the deglutathionylation activities
exhibited by these proteins. For instance, HsGSTO1-1 has been
shown to remove glutathione from S-(phenacyl)glutathione
(12), as this activity requires the presence of the catalytic Cys32
(13). In addition, GSTOs have been shown to be involved in the
reduction of methyl and dimethyl arsonate, an intermediate in
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table 1, Figs. 1 and 2, and additional references.
Theatomiccoordinatesandstructurefactors(code3PPU)havebeendepositedin
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence may be addressed: CRM2, UMR 7036 CNRS-UHP,
Nancy Universite´, Faculte´ des Sciences et Techniques, BP 70239, 54506
Vandoeuvre-les-Nancy Cedex, France. Tel.: 33-3-83-68-48-79; Fax: 33-3-83-
40-64-92; E-mail: Frederique.Favier@crm2.uhp-nancy.fr.
2 To whom correspondence may be addressed: UMR 1136 INRA-UHP, 3 The abbreviations used are: GSTO, glutathione transferase Omega; DHA, dehy-
IFR110, Nancy Universite´, Faculte´ des Sciences et Techniques, BP 70239,
54506 Vandoeuvre-les-Nancy, France. Tel.: 33-3-83-68-42-28; E-mail:
Eric.Gelhaye@lcb.uhp-nancy.fr.
droascorbate; GHR, S-glutathionyl-(chloro)hydroquinone reductase; HED,
hydroxyethyldisulfide;PDB, ProteinDataBank;PAP-SG, S-(phenacylacetophe-
none)glutathione; Se, selenium; CDNB, 1-chloro-2,4-dinitrobenzene.
9162 JOURNAL OF BIOLOGICAL CHEMISTRY
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GSTs of P. chrysosporium
the arsenic biotransformation (14, 15). Bacterial GSTOs, which
Expression and Purification of the Recombinant Proteins—
belong to subclass I, have been shown to be involved in penta- For protein production, the Escherichia coli BL21(DE3) strain
chlorophenol catabolism acting as S-glutathionyl-(chloro)hy- that contained the pSBET plasmid was co-transformed with the
droquinone reductase; this activity was also related to the different recombinant plasmids (20). Cultures were progres-
presence of a catalytic cysteine (16, 17). In fungi, few studies sively amplified up to 2.4 liters in LB medium supplemented
have been devoted to this GST class except in S. cerevisiae with ampicillin and kanamycin at 37 °C. Protein expression was
(ScGSTO). The three ScGSTOs belong to the subclass I and induced at exponential phase by adding 100 ␮M isopropyl ␤-D-
have been partially characterized particularly at the protein thiogalactopyranoside for 4 h at 37 °C. The cultures were cen-
level. The three proteins exhibit glutaredoxin-like activities trifuged for 15 min at 4400 ϫ g. The pellets were resuspended in
because they are able to reduce both hydroxyethyldisulfide and 30 ml of TE NaCl buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA,
dehydroascorbate in the presence of glutathione. In addition, 200 mM NaCl), and the suspension was stored at Ϫ20 °C.
the transcription of the three genes is induced by oxidative
Cell lysis was performed by sonication (three times for 1 min
stress (18, 19). Moreover, ScGSTO1 has been shown to be with intervals of 1 min), and the soluble and insoluble fractions
involved in sulfur metabolism and located in peroxisomes. were separated by centrifugation for 30 min at 27,000 ϫ g. The
PcGSTO1 and PcGSTO2, nevertheless, differ from their yeast soluble part was then fractionated with ammonium sulfate in
counterparts particularly at the H-site (the domain thought to two steps, and the protein fraction precipitated between 40 and
interact with the hydrophobic substrate).
80% of the saturation contained the recombinant protein, as
From phylogenetic analysis and biochemical analysis, Xun estimated by 15% SDS-PAGE. The protein was purified by size
et al. (17) recently proposed that the Omega subclass I mem- exclusion chromatography after loading on an ACA44 (5 ϫ 75
bers constitute a new class of GSTs named S-glutathionyl- cm) column equilibrated in TE NaCl buffer. The fractions that
(chloro)hydroquinone reductase (GHR) due to their activity contained the protein were pooled, dialyzed by ultrafiltration to
with S-glutathionyl-hydroquinones.
remove NaCl, and loaded onto a DEAE-cellulose column
The aim of this study was the investigation of the PcGSTOs, (Sigma) in TE buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA).
a class of GSTs that is over-represented in P. chrysosporium. The proteins were eluted using a 0–0.4 M NaCl gradient.
Recombinant proteins of isoforms that belong to the GSTO Finally, the fractions of interest were pooled, dialyzed, concen-
subclasses I and II have been biochemically characterized, trated by ultrafiltration under nitrogen pressure (YM10 mem-
exhibiting substrate specificity. Furthermore, the three-dimen- brane; Amicon), and stored in TE buffer at Ϫ20 °C. Purity was
sional structure of PcGSTO1 has been solved, allowing the first checked by SDS-PAGE. Protein concentrations were deter-
description of a S-glutathionyl-p-hydroquinone reductase at mined spectrophotometrically using a molar extinction coeffi-
Ϫ1
the structural level. From these biochemical and structural cient at 280 nm of 64,860 MϪ1ꢀcm for the PcGSTO1 WT,
data, the potential functions of the different isoforms are C86S, and 51340 MϪ1ꢀcmϪ1 for PcGSTO3 WT and C37S.
discussed.
E. coli strain B121 containing the pET-Pcgsto1 plasmid was
cultured as described previously (21) and was used as a source
of selenomethionine-substituted form of PcGSTO1 (SeMet-
EXPERIMENTAL PROCEDURES
Materials—Hydroxyethyldisulfide (HED) and 5,5Ј-dithiobis- PcGSTO1). The purification of SeMet-PcGSTO1 was per-
2-nitrobenzoic acid were from Aldrich and Pierce, respectively. formed following a procedure similar to that described for
All other reagents were from Sigma.
PcGSTO1.
Cloning and Construction of Pcgsto1 and Pcgsto3 Mutants by
Mass Spectrometry Analysis—PcGSTOs WT and mutated
Site-directed Mutagenesis—The open reading frame sequences were analyzed by quadrupole-TOF MS as described by Koh
encoding P. chrysosporium PcGSTO1 and PcGSTO3 were et al. (22).
amplified from P. chrysosporium cDNA using Pcgsto1 and
Fluorescence Properties of Wild-type and Mutated PcGSTOs—
Pcgsto3 forward and reverse primers (see supplemental Table 1) The fluorescence characteristics of PcGSTOs in the reduced
and cloned into the NcoI and BamHI restriction sites (under- and oxidized forms were recorded with a spectrofluorometer
lined in the primers) of pET3d (Novagen). For PcGSTO3, the (Cary Eclipse; VARIAN) in TE buffer at a protein concentration
amplified sequence encoded a protein in which an alanine has of 10 ␮M.
been added during cloning. Using two complementary muta-
Determination of Free Thiol Groups—The number of free
genic primers, the two cysteines of PcGSTO3 were individually thiol groups in either untreated, denatured, or reduced condi-
substituted into serines, and the primers are listed in supple- tions was determined spectrophotometrically with 5,5Ј-dithio-
mental Table 1. For PcGSTO1, due to the presence of the signal bis-2-nitrobenzoic, as described by Koh et al. (22). All thiol
peptide, a first construction has been made, and the resulting titrations were performed on enzymes either as purified or after
protein starts with the N-terminal sequence MATTICLRH. SDS treatment or after dithiothreitol (DTT) reduction. In the
Because we obtained a mixture of different proteins after pro- latter case, proteins were first reduced by incubation with 10
duction and purification (see under “Results”), a new construc- mM DTT for 30 min at room temperature, then precipitated
tion has been made. The resulting protein with an additional with 1 volume of 20% (w/v) trichloroacetic acid (TCA), and
alanine started thus with the N-terminal sequence MASFT- finally stored on ice for 30 min. The mixture was then centri-
TGST (see supplemental Fig. 1). PcGSTO1 used for structure fuged for 10 min at 13,000 ϫ g, and the pellets were washed
determination consists also of the 352 last residues of the full- twice with 2% (w/v) TCA. The precipitate was resuspended in
length protein.
30 mM Tris-HCl, pH 7.0, 1 mM EDTA, and 1% (w/v) SDS. The
MARCH 18, 2011•VOLUME 286•NUMBER 11
JOURNAL OF BIOLOGICAL CHEMISTRY 9163

GSTs of P. chrysosporium
TABLE 1
concentration was estimated by UV spectrophotometry. For
denatured proteins, samples were incubated in the presence of
2% SDS treatment during 30 min before 5,5Ј-dithiobis-2-nitro-
benzoic addition.
Statistics of X-ray diffraction data collection and model refinement
Data collection
Space group
Cell dimensions
Resolution
Rmerge
C2
a, 166.8 Å, b, 70.3 Å, c, 72.6 Å; ␤, 98.8°
46.66 to 2.30 Å (2.43 to 2.30 Å)a
Activity Measurements—The activity measurements of WT
or mutant PcGSTOs in the HED assay or for reduction of dehy-
droascorbate (DHA) were performed as described by Couturier
et al. (23). Dimethylarsinate reductase and glutathionyl-phenyl-
acetophenone assay were performed as described previously
(12, 13). The GST activity was measured with 0.2 mM CDNB as
the substrate in 30 mM Tris buffer, pH 8.0, and the continuous
absorbance at 340 nm was monitored. S-Glutathionyl-p-hydro-
quinone reductase activity was monitored as follows: 1 mM 1,4-
benzoquinone in 30 mM Tris-HCl, pH 8, was used as base line
for an absorbance spectrum (230–400 nm). After 2 min, 1 mM
reduced glutathione (GSH) was added to the reaction mixture,
and the spectra were monitored every minute during 5 min. At
last, purified recombinant proteins were added in the reaction
mixture, and spectra were recorded every minute. In comple-
mentary experiments, 1 mM 1,4-benzoquinone in 30 mM Tris-
HCl, pH 8, was incubated in the presence of 1 mM GSH, 200 ␮M
NADPH, 0.5 IU glutathione reductase before the addition of
the protein of interest. The activity was followed by monitoring
3.7% (18.8%)
19.8 (4.8)
98.9% (96.0%)
4.3
I/␴I
Completeness
Redundancy
Refinement
Resolution
46.66 to 2.30 Å
36,622
No. of reflections
b
R
all/Rfree
19.2/22.8
No. of atoms
Protein
2572 (monomer A) ϩ 2451 (monomer B)
1 glutathione (20 atoms) in monomer A
223
Ligand
Water
B-factors
Protein
37.9 (monomer A); 41.6 (monomer B)
Ligand
49.4
38.6
Water
Ramachandran statistics
Residues in preferred regions
Residues in allowed regions
Outlier residues
Root mean square deviations
Bond lengths
Bond angles
95.7%
4.3%
0.0%
0.011 Å
1.25°
a Values in parentheses are for highest resolution shell.
b Rall is determined from all the reflections (working set ϩ test set), and Rfree cor-
responds to a subset of 5% of reflections (test set).
the decrease in absorbance arising from NADPH oxidation in ␮l of the PcGSTO1 solution at various concentrations and 1.5
this coupled enzyme assay system that showed the formation of ␮l of various crystallization solutions were deposited in a
glutathione disulfide (GSSG).
72-well Microbatch plate (Hampton Research) pre-filled with
In complementary experiments, S-(phenacylacetophenone)- paraffin oil and stored at 4 °C. Preliminary results have been
glutathione (PAP-SG) and 2-methyl-S-glutathionyl-naphtho- obtained from two commercial screening solutions as follows:
quinone (thiodione) were prepared as described previously (24, the condition B2 from the JBScreen classic kit 2 (Jena Biosci-
25), and the purity and the nature of the products were verified ence) and the Crystal Screen solution 6 from Hampton
by mass spectrometry. Activities of the WT and variant Research. Further trials aimed to improve reproducibility and
enzymes were assayed by following the appearance of degluta- crystal quality gave the best results for PcGSTO1 solutions at
Ϫ1
thionylated menadione (at 425 nm) and phenylacetophenone 30–40 mgꢀml
and crystallization solutions composed of
(at 290 nm). The reactions were performed in 30 mM Tris-HCl, 20–30% polyethylene glycol 4000 (PEG 4000), 0 to 0.2 M mag-
pH 8, containing GSH (5 mM) and substrates (from 15 to 2 mM) nesium or calcium chloride, and 0.1 M Tris-HCl, pH 8.5. How-
in a final volume of 650 ␮l and initiated by adding WT or ever, polycrystals grew frequently so that only rare portions of
mutant enzyme. The concentration of enzyme added was them were suitable for x-ray data collection. No crystal of non-
adjusted based on preliminary activity experiments, ranging selenomethionylated PcGSTO1 suitable for data collection has
from 0.1 and 1 ␮M. The reactions were stopped by adding 350 ␮l been obtained.
of ethanol, then strongly mixed by vortex, then centrifuged at
The diffraction data were collected at 100 K on the syn-
14,500 rpm during 15 min, and analyzed by HPLC using a Gem- chrotron beamline FIP-BM30A (26) at ESRF (France) with a
ini 5-␮m C18 column (150 mm long ϫ 4.6 mm inner diameter) wavelength of 0.9805 Å corresponding to the peak of the Se
Ϫ1
at 22 °C.
K-edge, from a crystal grown with 40 mgꢀml PcGSTO1,
To visualize the deglutathionylation reactions, PAP-SG or 25% PEG 4000, and no salt, and flash-frozen after a quick
thiodone (15 ␮M) was added in a 650-␮l reaction mixture con- immersion in the crystallization solution mixed with 20%
taining 30 mM Tris-HCl, pH 8, and 15 ␮M enzyme. After stop- glycerol. The data sets were processed with XDS (27) and
ping the reaction by adding ethanol, samples were analyzed by scaled with SCALA from the CCP4 Package (28). Statistics
HPLC as described above.
are summarized in Table 1.
Size Exclusion Chromatography—Size exclusion chromatog-
Structure Solution and Quality of the Model—The PcGSTO1
raphy experiments have been performed using a Superose structure was solved using the single wavelength anomalous
12HR column connected to a fast protein liquid chromatogra- dispersion protocol of Auto-Rickshaw at the EMBL-Hamburg
phy (FPLC) system. The column was equilibrated with 30 mM automated crystal structure determination platform (29). The
Tris-HCl buffer, pH 8, containing 0.15 M NaCl, and the proteins input diffraction data were prepared and converted for use in
Ϫ1
were eluted in the same buffer at a flow rate of 0.25 mlꢀmin
.
Auto-Rickshaw using programs of the CCP4 suite (28). Nine
Crystallization and X-ray Diffraction Data Collection—Prior heavy atoms among the 10 requested were found using the pro-
to crystallization, protein samples were concentrated to 40 gram SHELXD (30). Initial phases were calculated after density
Ϫ1
mgꢀml
PcGSTO1, in TE buffer. Crystals of the SeMet- modification using the program SHELXE (31), at 3.0 Å resolu-
PcGSTO1 were obtained by using the microbatch method. 1.5 tion. The initial phases were improved using density modifica-
9164 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 11•MARCH 18, 2011

GSTs of P. chrysosporium
tion and phase extension to 2.30 Å resolution using the pro- three and two thiols per protein were titrated for the reduced
gram DM (32). 70% of the model was built by ARP/wARP (33, and denatured PcGSTO1 WT and PcGSTO3 WT proteins,
34). It was progressively completed and refined by using itera- respectively, in accordance with the expected thiol content.
tively COOT (35) and REFMAC5 (36), including diffraction Under denaturing conditions and after reduction, the obtained
data up to 2.3 Å resolution. An electron density further attrib- values for PcGSTO1 C86S (around two thiols per protein) and
uted to one GSH bound in the active site appeared in monomer PcGSTO3 C37S (around one thiol per protein) were in agree-
A during refinement, although no GSH was added to crystallize ment with the theoretical values. Comparison of values
PcGSTO1. On the contrary, the corresponding area of mono- obtained for denatured PcGSTO1 WT and C86S suggested that
mer B only displayed a few poor spherical densities that more the sulfur of Cys86 was involved at least partially in a DTT-
probably corresponded to water molecules. Final model statis- reducible link. For PcGSTO3 WT, one cysteinyl residue was
tics are shown in Table 1.
probably involved in a disulfide bridge, whereas for PcGSTO3
Most residues corresponded to well defined electron density, C37S, one additional thiol group seemed to be noncovalently
except for the N-terminal part of both monomers that appeared bound in the protein.
too disordered to be observed. Thus, models of monomers A
Fluorescence Spectrometry and Mass Analysis—PcGSTO3
and B contained residues Glu38–Asp351 and residues Phe54– contains two tryptophans adjacent to the active site. Given that
Asp351, respectively. Furthermore, the residues Glu38–Ser53 proximity, we have investigated whether the intrinsic fluores-
only observed in monomer A displayed a weaker electron den- cence of PcGSTO3 could change under reducing (DTT) or oxi-
sity and higher B factors with respect to the rest of the structure. dizing (GSSG or diamide) conditions (Fig. 2). The native pro-
Figs. 4 and 5 were prepared by using PyMOL.
tein displayed an emission spectrum with a maximum at 350
nm characteristic of a fluorescence signal strongly dominated
by Trp. Adding 400 ␮M DTT to native PcGSTO3 immediately
RESULTS
PcGSTO1 belongs to the first subclass of GSTs Omega dis- led to a strong increase of the fluorescence signal (Fig. 2). The
playing a CPWATR potential active site compatible with the addition of GSSG (10 mM) on previously DTT-reduced PcG-
CP(W/F)(A/T)(H/Q)R characteristic of the members of this STO3 strongly decreased the fluorescence signal, whereas addi-
subclass (Fig. 1, top and bottom) (9). According to the predic- tion of diamide (10 mM) did not change the reduced PcGSTO3
tion softwares, PcGSTO1 possesses a signal peptide targeting to fluorescence spectrum (data not shown). Similar experiments
mitochondria. Different attempts to produce heterologously have been performed with PcGSTO3 C37S; however, in this
the full-length protein in E. coli resulted in a mixture of trun- case no changes in the fluorescence spectrum have been
cated proteins and suggested that this N-terminal extension obtained whatever the reductant and oxidant used. In accord-
contained putative cleavage sites recognized by the E. coli ance with thiol titrations, these data suggested that the Cys37 of
machinery or an intrinsic instability of the precursor. These data the native PcGSTO3 WT was glutathionylated and that the
were in accordance with the presence of a signal peptide in the tryptophan environment strongly changed after glutathionyla-
N-terminal part of the protein as shown previously for different tion of this active site cysteine.
other redox proteins (37, 38). To obtain a homogeneous protein
To confirm this hypothesis, PcGSTO3 WT and PcGSTO3
preparation, a shorter form of PcGSTO1 was produced and puri- C37S were analyzed by mass spectrometry. A Q-TOF analysis
fied removing the N-terminal extension of the protein (46 amino of the DTT-reduced PcGSTO3 revealed a single protein with a
acids). Nevertheless, the measured enzymatic activities and speci- molecular mass of 29,615 Da, which was consistent with a pro-
ficities (full-length and without signal peptide) described in this tein where the N-terminal methionine was cleaved. After treat-
study were similar for both forms (data not shown).
ment with GSSG, the mass of the protein increased by around
PcGSTO3 belongs to the subclass II of the GSTs Omega with 305 Da, a feature consistent with the formation of one glutathi-
an FCPFVQ active site quite similar to the FCPFAE of the one adduct. This obtained value of 29,921 Da also corresponded
HsGSTO1-1 (Fig. 1, bottom). A second cysteinyl residue is pres- to the mass of the native protein, and thus confirmed this latter
ent at the same position into both proteins (Cys94, PcGSTO3 also possessed a glutathione adduct on Cys37. For the mutant
numbering). Different attempts have been made to produce PcGSTO3 C37S, the analysis revealed a single protein with a
heterologously the mutated protein PcGSTO3 C94S, but they molecular mass of 29,599 Da, which was consistent with a pro-
remained unsuccessful because the resulting protein was fully tein without the first methionine and which harbored the Cys
insoluble.
to Ser mutation. This result combined with the thiol titration
Thiol Content Determination—To investigate the putative was in agreement with the noncovalent binding of one reduced
role of the conserved cysteines in the catalytic mechanism of glutathione to PcGSTO C37S.
both proteins, site-directed mutagenesis has been used to pro-
Similar experiments (fluorescence and mass spectrometry)
duce the recombinant proteins in E. coli, PcGSTO1 C86S and have been conducted with PcGSTO1 and PcGSTO1 C86S. Even
PcGSTO3 C37S. The thiol content of these proteins has been when a tryptophan residue was present near the putative catalytic
determined in different conditions as follows: native oxidized cysteine, the fluorescence spectrum remained unchanged what-
corresponding to nonreducing and nondenaturing conditions; ever the reductant (GSH/DTT) or the oxidant (GSSG/diamide)
denatured oxidized corresponding to a titration in the presence used. A Q-TOF analysis of the DTT-reduced protein revealed a
of SDS; reduced and denatured corresponding to a reduction single protein with a molecular mass of 40,187 Da, which was con-
followed by a protein precipitation (see “Experimental Proce- sistent with a protein where the methionine was cleaved. After
dures”). A summary of these data is shown in Table 2. Nearly treatment with GSSG, the mass of the reduced protein increased
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GSTs of P. chrysosporium
FIGURE 1. Alignment of PcGSTO1 (top) and PcGSTO3 (bottom) with homologs. Amino acids involved in the catalytic site are labeled. NCBI accession
numbers are as follows: PcGSTO1 (EU791894), PcGSTO3 (EU 791893), PcGSTO4 (JGI accession number 7168), Sphingobium chlorophenolicum PcpF (AAM
96671), Escherichia coli YqjG (NP_417573), and Homo sapiens GSTO1–1 NP_004823. For PcGSTO1, the putative signal peptide is in italic.
by around 306 Da, a feature consistent with the formation of one
TABLE 2
glutathione adduct. This obtained value of 40,493 Da corre-
Number of free thiols in WT and mutated PcGSTOs under various
redox conditions
The native column is indicative of the protein thiol content measured after purifi-
cation. Proteins were denatured using SDS treatment or reduced by DTT and sub-
sequently precipitated using TCA. The thiol content per protein was quantified by
5,5-dithiobis(nitrobenzoic acid). Data are represented as mean Ϯ S.D. (n Ϯ 4).
sponded also to the mass of the native protein and confirmed this
latter also possessed a glutathione adduct. These data combined
with thiol titration results were consistent with the potential glu-
tathionylation of Cys86, because no glutathione adduct was
observed on PcGSTO1 C86S.
Substrate Specificity between PcGSTO1 and PcGSTO3—Both
PcGSTOs were found to be active in the two classical glutare-
doxin assays as follows: the reduction of DHA and HED assays.
Native
Denatured Reduced and denatured
PcGSTO1
PcGSTO3
0.46 Ϯ 0.12 2.50 Ϯ 0.25 3.0 Ϯ 0.2
PcGSTO1 C86S 0.15 Ϯ 0.03 2.0 Ϯ 0.1 2.04 Ϯ 0.12
0.15 Ϯ 0.03 1.00 Ϯ 0.03 1.80 Ϯ 0.13
PcGSTO3 C37S 0.92 Ϯ 0.09 1.78 Ϯ 0.12 1.00 Ϯ 0.04
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GSTs of P. chrysosporium
The activity of the PcGSTOs in these two assays increased lin- shown). To determine the catalytic parameters, PAP-SG was
early with increasing protein concentrations in the 0–750 and synthesized and purified, and the activity against this substrate
0–100 nM concentration ranges, respectively. As expected, was followed by analytical HPLC in the presence of GSH (Table
these observed “thiol transferase” activities are related to the 3). In these conditions, only PcGSTO3 is active with a catalytic
Ϫ1
presence of a cysteine in their catalytic site, because the efficiency of around 4ꢀ105
M
s
Ϫ1, a value greater than that
obtained for HsGSTO1-1 (104
M
Ϫ1 sϪ1) (39). The incubation of
mutants PcGSTO1 C86S and PcGSTO3 C37S are fully inactive
in these assays (data not shown). The kinetic analyses revealed
catalytic efficiency values (kcat/Km) similar between both
PcGSTOs against DHA, whereas PcGSTO1 exhibited a slightly
stronger activity against HED than PcGSTO3 (Table 3). Inter-
estingly, whereas PcGSTO1 C86S remained inactive in all
tested assays, PcGSTO3 C37S exhibited a classical glutathione
transferase activity detected using CDNB as the substrate. Both
PcGSTOs WT were fully inactive against CDNB.
previously reduced PcGSTO3 in the presence of PAP-SG with-
out addition of GSH led to the formation of phenacylaceto-
phone detected by analytical HPLC (supplemental Fig. 2A).
This deglutathionylated product is not observed either in the
presence of native purified protein (i.e. with a glutathione
adduct) or in the presence of PcGSTO3 C37S. These results are
in accordance with the involvement of Cys37 in the deglutathio-
nylation activity leading to the formation of a disulfide bridge
with the glutathione removed from PAP-SG. This hypothesis
was confirmed by mass spectrometry analysis of the resulting
protein showing the presence of a glutathione adduct (data not
shown).
The HsGSTO1.1, ortholog of PcGSTO3 has been shown to
possess activity against various substrates, including dimethy-
larsinate and 2-bromo-4Ј-phenylacetophenone (12, 13). In
both cases, a glutathione adduct occurred followed by a deglu-
tathionylation reaction catalyzed by the enzyme, preventing
access to the initial substrate concentrations and also to kinetic
parameters. As expected, PcGSTO3 was active in these tests in
the presence of glutathione, whereas PcGSTO3 C37S, PcG-
STO1 WT, and PcGSTO1 C86S were fully inactive (data not
In additional experiments, we tested quinones previously
incubated with reduced glutathione as substrates for PcGSTOs.
In these conditions, Michael addition occurred leading to the
formation of glutathione adducts corresponding to S-glutathio-
nyl-p-hydroquinones (40, 41). As shown in Fig. 3, addition of
reduced glutathione strongly modified the absorbance spec-
trum of 1,4-benzoquinone with the appearance of a peak max-
imum at 305 nm in accordance with the formation of S-gluta-
thionyl-p-benzohydroquinone as described previously (42).
Addition of PcGSTO1 led to a strong modification of the
absorbance spectrum, with the disappearance of the 305-nm
peak and the formation of a peak at 290 nm corresponding to
p-benzohydroquinone. In contrast, no effect was observed in
the presence of PcGSTO1 C86S and PcGSTO3 (data not
shown). All these data were consistent with a deglutathionyla-
tion reaction of S-glutathionyl-p-benzohydroquinone. As ex-
pected, the activity of PcGSTO1 against S-glutathionyl-p-ben-
zohydroquinone led to the production of GSSG as revealed by a
coupled enzymatic test performed using glutathione reductase
(data not shown). Similar data were obtained using menadione
(2-methyl-1,4-naphthoquinone), the glutathionylated product
(S-glutathionyl-naphthoquinone) being characterized by an
absorbance peak at 425 nm (data not shown) (43). The obtained
results suggested a preference of PcGSTO1 for aromatic sub-
strates in contrast to PcGSTO3. To obtain catalytic parameters,
FIGURE 2. Fluorescence spectra of PcGSTO3 under different redox states.
Emission spectra of native (ꢁ), DTT-reduced (f), and subsequently GSSG-
oxidized (F) PcGSTO3 (excitation at 290 nm) were recorded with 10 ␮M pro-
tein at 25 °C in TE buffer, pH 8.0,. The reduced PcGSTO3 was obtained by a
treatment of 10 ␮M protein with 400 ␮M DTT. PcGSTO3 was oxidized using 10
mM GSSG after DTT reduction.
TABLE 3
Kinetic parameters of PcGSTO1 and PcGSTO3 in HED, DHA, and CDNB activity assays
The apparent Km value for GSH in the DHA assay was determined using a GSH concentration range of 0.2–4 mM in the presence of 2 mM DHA for PcGSTO1 and
PcGSTO3. The apparent Km value for GSH was determined for PcGSTO3 C37S in the CDNB assay using a GSH concentration range of 0.1–2 mM in the presence of 2 mM
CDNB. The apparent Km value for DHA was determined using a concentration range of 0.1–4 mM in the presence of 3 mM GSH. The apparent turnover values (kcat) for HED
were determined in the presence of 4 mM GSH and 4 mM HED. The apparent Km value for CDNB was determined using a concentration range of 0.1–2 mM in the presence
of 2 mM GSH. Concerning PAP-SG and thiodione, the apparent Km values were determined using a substrate concentration range of 0.015–4 mM. The apparent Km and kcat
values were calculated by nonlinear regression using the Michaelis-Menten equation. Data are represented as mean Ϯ S.D. (n Ϯ 3). ND means not detected. The detection
limit was estimated at 0.5 mIU for spectrophotometric tests, whereas 0.5 nmol of either menadione or phenacylacetophenone was detected using analytical HPLC.
GSH
HED
DHA
CDNB
PAP-SG
Thiodione
K
m (mM)
PcGSTO1
PcGSTO3
PcGSTO3 C37S
؊1
1.65 Ϯ 0.33
1.44 Ϯ 0.25
0.18 Ϯ 0.04
1.84 Ϯ 0.40
0.33 Ϯ 0.07
ND
ND
2.00 Ϯ 0.62
ND
ND
0.47 Ϯ 0.19
0.97 Ϯ 0.27
ND
ND
k
cat (s
)
PcGSTO1
PcGSTO3
PcGSTO3 C37S
2.67 Ϯ 0.01
15.75 Ϯ 0.26
ND
1.81 Ϯ 0.17
0.97 Ϯ 0.06
ND
ND
19.75 Ϯ 2.31
ND
19.01 Ϯ 2.89
ND
18.43 Ϯ 1.90
ND
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GSTs of P. chrysosporium
PcGSTO1 adopts the canonical GST fold composed of an
N-terminal thioredoxin-like domain and a C-terminal ␣-helix
domain. It also possesses several additional features as follows:
a long N-terminal coil of 77 residues; a loop that connects ␤2 to
␣2 elongated by 20 residues compared with typical GSTs; and a
C-terminal extension corresponding to a ninth ␣-helix fol-
lowed by a coil of 20 residues (Fig. 4 and supplemental Fig. 1).
This helix ␣9 (Phe319 to Ile333) is the only secondary structure
among these supplementary features. It adds to the ␣-helix
domain, near the C-terminal end of helix ␣6, so that it also
interacts with the thioredoxin-like domain via the short loop
that connects the strand ␤1 and the ␣-helix ␣1 and via the long
loop that goes from ␤2 to ␣2. Helix ␣9 is a structural character-
istic of GST Omega (10) and is also present in GST Tau (45) and
GST Delta (46). In these GSTs, the few residues that follow ␣9
(when they exist) go toward the thioredoxin-like domain to
interact with the loops ␤1-␣1 and ␤2-␣2. In PcGSTO1, the long
loop observed between ␤2 and ␣2 hinders this interaction, so
the 20 residues that follow ␣9 in PcGSTO1 form a coil that runs
at the opposite side of the thioredoxin-like domain, antiparallel
to ␣9.
FIGURE 3. PcGSTO1 activity against S-glutathionyl-p-benzohydroqui-
none. Absorption spectra have been recorded adding successively in 1 ml at
25 °C in TE buffer, pH 8.0, 1 ␮M p-benzoquinone (ꢁ), 5 ␮M reduced GSH (f),
and 0.28 ␮M PcGSTO1 (ꢁ). In a separate experiment, as control, absorption
spectrum of 1 ␮M p-benzohydroquinone was recorded at 25 °C in TE buffer,
pH 8.0.
S-glutathionyl-naphthoquinone, also called thiodione, was syn-
This C-terminal coil allows the formation of a dimer that
thesized and purified, and the activity against this substrate was completely differs from the usual GST dimer, where the two
followed by analytical HPLC (Table 3). The activity of PcG- PcGSTO1 monomers associate exclusively via their ␣-helix
STO1 against thiodione leads to the formation of a product that domains (Fig. 4), with a large buried area (3100 Å2) as deter-
elutes with the same retention time as menadione (data not mined by PISA (44). After ␣9, the 20 C-terminal residues of one
shown). The kcat value with this substrate is around 10-fold monomer mainly interact with residues of the helix ␣5 N-ter-
higher than the values obtained with HED and DHA. No activ- minal end (H-bonds to the side chains of Gln231 and Tyr234),
ity against thiodione was detected using PcGSTO1 C86S, con- next with a residue of the ␣6 C-terminal end (Thr283), and then
firming the importance of the cysteinyl residue in the catalysis with a residue of the loop that follows (Asn288), in the other
of the deglutathionylation reaction. In additional experiments, monomer. These direct hydrogen bonds only concern the main
no glutathione was added in a mixture containing PcGSTO1 chain atoms of the C-terminal end and add to several hydro-
and thiodone, the results of the reaction being analyzed by phobic contacts. Is this dimer a crystal artifact or a physiological
HPLC (supplemental Fig. 2B). In these conditions, addition of entity? The large surface of contact between the protomers and
reduced PcGSTO1 led to the formation of menadione. Analysis the involvement of the C-terminal coil, specific to PcGSTO1
by mass spectrometry of the resulting protein showed the pres- and related proteins, in the inter-monomer interactions argue
ence of a glutathione adduct (data not shown). No menadione for a physiological dimer, as do the dynamic light scattering and
formation was observed when PcGSTO1 C86S was used.
size exclusion chromatography results. Furthermore, this
Dimerization—Size exclusion chromatography and dynamic dimer, different from the canonical GST dimer, is similar to the
light scattering were used to determine the oligomeric nature of dimer assigned by Cuff et al. (Midwest Center for Structural
the native forms of PcGSTO1 and PcGSTO3. The retention Genomics) in the crystal structure of a putative glutathione
times of the size exclusion chromatography allowed estimation S-transferase from Corynebacterium glutamicum recently
of the following native molecular masses: ϳ85,000 Da for PcG- released by the Protein Data Dank (PDB code 3M1G).4 The
STO1 and ϳ62,000 Da for PcGSTO3. Similar results have been similar dimerization mode appears clearly related to a mono-
obtained using dynamic light scattering suggesting that both mer structure found by DALI (47) to be the closest to the PcG-
enzymes were dimers.
STO1 structure (root mean square deviation, 1.092 Å for 233
Crystal Structure of PcGSTO1—The structure of PcGSTO1 superimposed residues).
has been solved to 2.3 Å resolution by single wavelength anom-
PcGSTO1 also possesses an N-terminal extension with
alous dispersion using crystals of SeMet-PcGSTO1 (Table 1). respect to other GSTs, by far much longer than the 20 residues
The crystals belonged to the space group C2, and the asymmet- observed in GSTOs, because 77 residues precede the entrance
ric unit consisted of two polypeptide chains (monomer A, res- to ␤1. No electron density was observed for the 37 first residues,
idues Glu38–Asp351; monomer B, residues Phe54–Asp351; root which were thus supposed to be highly disordered. Residues
mean square deviation, 0.348 Å for 298 superimposed C␣ Glu38 to Ser53 were only observed in monomer A (see under
atoms), one GSH molecule not covalently bound and 223 water “Experimental Procedures”). The main chain of residues 38–53
molecules. Crystal packing analysis using PISA (44) suggested first runs between the long loop that connects ␤2 to ␣2 and the
that the asymmetric unit content corresponded to a dimer. The
two protomers are related to each other by a noncrystallo-
graphic 2-fold symmetry axis.
4 M. E. Cuff, N. Marshall, G. Cobb, and A. Joachimiak, unpublished results.
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GSTs of P. chrysosporium
FIGURE 4. Schematic drawing of the PcGSTO1 (left) and HsGSTO1-1 (10) (right) structures. The monomers are displayed at the top separated by their
common topology diagram mentioning the secondary structure numbering; the dimers are shown at the bottom, with orientations chosen to render their
assembling at most. In each case, the thioredoxin-like domain is colored in cyan, and the ␣-helix domain is in purple. In monomers, the other colors emphasize
the features that distinguish PcGSTO1 from HsGSTO1-1 as follows: the N-terminal coil in orange, the loop that connects the strand ␤2 to the ␣-helix ␣2 in blue,
and the C-terminal coil in black. The gray sticks correspond to glutathione, only present in one monomer of PcGSTO1 and in the two monomers of HsGSTO1-1.
loop that goes from ␣2 to ␤3 in the N-terminal thioredoxin-like particular, the GSH S␥2 atom lies at 3.25 Å from the Tyr223 OH
domain. Then it interacts with the short loop between ␣4 and group tightly hydrogen-bonded to the Tyr326 OH.
␣5 in the ␣-helix domain, where it forms a turn before it enters
In addition to the usual interactions with the main chain of a
the region Phe54–Arg77 observed in both monomers. This seg- residue (Val158) that precedes the conserved cis-proline at the
ment adopts an extended conformation to return toward the entrance of the strand ␤3, the GSH cysteine main chain forms
N-terminal domain. There it interacts with the loops that con- an additional hydrogen bond between its CO group and the
nect ␣2 to ␤3 and ␤4 to ␣3, before it runs all along ␣3, parallel to N⑀1 atom of Trp119, situated in the long loop between ␤2 and
it. Then it enters ␤1. The conformation of the PcGSTO1 N-ter- ␣2 absent in most GSTs (Fig. 5). In the same way, besides the
minal end confers it with the original property to form part of usual stabilization of the GSH glutamyl residue (main chain by
the active site, composed of the G-site for GSH binding and the residues Glu173 and Ser174 from the loop between ␤4 and ␣3
H-site, which receives the hydrophobic part of the glutathio- and aliphatic part of the side chain by hydrophobic contact with
nylated substrate.
Trp88 from the N-terminal end of ␣1), an additional hydrogen
The G-site—Only monomer A displayed an electron density bond was observed between the GSH glutamyl atom O⑀1 and
consistent with modeling of a GSH molecule (see under “Exper- the N␩1 atom of Arg56 in the long N-terminal extension of
imental Procedures”). However, no major structural difference PcGSTO1. Finally, the oxygen atoms of the carboxylic group of
between monomers A and B was observed that could account the GSH glycyl residue form two hydrogen bonds with the side
for an asymmetric binding of GSH. Nevertheless, in this struc- chain of Arg155 situated in the loop between ␣2 and ␤3 slightly
ture only residues that belong to monomer A contributed to longer than in the other GSTs (Fig. 5), although usually only one
GSH binding.
bond is observed with a basic residue (48).
No disulfide bridge exists between the GSH cysteine side
To summarize, some of the additional features that PcG-
chain and the catalytic residue Cys86 situated at the N-terminal STO1 displays with respect to other GSTs (the long N-terminal
end of helix ␣1 (Fig. 5). The sulfur to sulfur distance is 3.9 Å; end, the long loop between ␣2 and ␤2, and the few supplemen-
the electron density is unambiguous, and careful analysis of tary residues between ␣2 and ␤3) contribute to GSH binding.
the diffraction data reveals no radiation damage that could have They squeeze GSH so much that only 45 Å2 of its surface is
disrupted a pre-existing disulfide bond. Superposition of the accessible to solvent (Fig. 5). As a comparison, this value is 148
structures of PcGSTO1 and HsGSTO1-1 (where a disulfide Å2 in HsGSTO1.1. The active site seems so enclosed that it
bridge exists) shows a conserved orientation of the catalytic raises the question of its accessibility not only to the GSH part
cysteine side chain. On the contrary, a displacement of the GSH of the glutathionylated substrate but also to its hydrophobic
S␥2 atom is observed in PcGSTO1, which results from slight part.
differences in the rotation angles around the bond between C␥1
The Putative H-site—The lack of a structure of PcGSTO1
and C␦1 in the GSH glutamyl residue and around the bond with a bound S-glutathionylated substrate forbids direct exper-
between C␣2 and C␤2 in the GSH cysteinyl side chain. The imental delineation of the H-site. The PcGSTO1 surface shows
latter points toward three tyrosine residues (Fig. 5), the no clear accessible cavity that could accommodate the hydro-
hydroxyl groups of which form a hydrogen bond network; in phobic part of the aromatic S-glutathionylated substrate (Fig.
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GSTs of P. chrysosporium
FIGURE 5. Substrate-binding sites of PcGSTO1 and HsGSTO1-1 (10). Top, stereo view of the hydrogen bond network formed by the active site residues
aroundGSHinPcGSTO1. Theschematictraceofthepolypeptidemainchainissuggestedin cyan(thioredoxin-likedomain)andpurple(␣-helixdomain). Middle,
PcGSTO1 electrostatic surface (blue, positive charge; red, negative charge) of PcGSTO1 in the region of GSH (shown as sticks). Left panel, calculated with all the
residues observed for monomer A in the crystallographic model (Glu38–Asp351); middle panel, calculated without the N-terminal coil (i.e. with residues
Tyr78–Asp351); right panel, same as previous but without Arg155. Bottom, HsGSTO1-1 active site compared with PcGSTO1. Left panel, electrostatic surface
calculated for the HsGSTO1-1dimer, in the region of GSH (shown as sticks). Right panel, stereo view of the accessible surface of HsGSTO1-1 (gray) in the region
of GSH (green), superimposed to the PcGSTO1 active site containing the GSH (blue sticks). Residues Tyr223, Tyr326, and His330 fill the pocket defined by Board et
al. (10) as the HsGSTO1-1 active site. Residues in cyan and in orange form a second and a third layers of residues that almost close the PcGSTO1 active site.
5). Comparison with HsGSTO1-1 emphasizes the absence of a and Alpha from chicken (PDB code 1VF3).5 In each of these
structures, 1-(S-glutathionyl)-(2,4-dinitrobenzene) leans its
benzene ring against the hydrophobic side chains of the resi-
dues that belong to the ␣4 C-terminal end, and it often interacts
with the first N-terminal residue of ␣1 (using PcGSTO1 num-
bering of secondary structures). Comparable interactions could
be obtained using Trp88, Tyr223, and Phe227, but they would
require a slightly different positioning of the 1-(S-glutathionyl)-
(2,4-dinitrobenzene) glutathionyl part with respect to the cur-
rent position of GSH in PcGSTO1. Indeed, in the structure,
GSH appears too shifted toward the ␣-helix domain, with its
cysteinyl sulfur atom exactly aligned on the ␣1 axis. The current
position of the long N-terminal loop shows that residues such
as Met46, Phe54, or Arg56 could also be involved, but once again,
large binding pocket; the bottom of the HsGSTO1-1 putative
H-site (formed by Phe31, Cys32, Pro33, Arg183, Trp222, and
nearby residues (10)) is filled, in PcGSTO1, by Tyr223 and
Tyr326, which surround the GSH cysteinyl side chain, and by
Arg56, Cys86, and His325 (Figs. 1 and 5). Ser44, Met46, Phe54,
Lys57, Arg155, Phe227, and His330 form a narrow, deep cylindri-
cal hole, incompatible with access and binding of a bulky
substrate.
Searching in the PDB for X-ray structures of GSTs in com-
plex with S-glutathionylated substrates, 1-(S-glutathionyl)-
(2,4-dinitrobenzene) was found to be the closest analog to
S-glutathionyl-p-benzohydroquinone. It has been used as a
substrate by the GSTs of the classes Mu from rat (PDB code
5GST (49)) and human (PDB code 1XWK (50)), Pi from human
(PDB code 18GS (51), Sigma from squid (PDB code 1GSQ (52)), 5 S. C. Lin, Y. C. Lo, M. F. Tam, and Y. C. Law, unpublished results.
9170 JOURNAL OF BIOLOGICAL CHEMISTRY
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GSTs of P. chrysosporium
the main question concerns access of the substrate to its bind- rapidly at the beginning of the radiation exposure because no
ing site, which would likely require a movement of the N-ter- residual electron density corresponding to this bond was
minal loop.
observed and the sulfur to sulfur distance is large (3.9 Å), con-
trary to documented cases where such a phenomenon has been
described. At the moment, the reasons for the discrepancy
DISCUSSION
Compared with other organisms, the GSTO class of enzymes between the PcGSTO1 x-ray structure and the results of thiol
is considerably larger in saprotrophic fungi and particularly in titrations concerning the presence of a disulfide bond remain to
P. chrysosporium. From primary structure homology, this class be elucidated.
could be divided in three different subclasses in fungi (9), one
As defined by Xun et al. (17), the main biochemical charac-
related to plant Lambda GSTs and bacterial GSTs, whereas the teristic of GHRs is the ability to remove glutathione from S-glu-
second and the third subclasses are related to animal isoforms. tathionyl-(chloro)hydroquinone substrates. In this study, we
In accordance with our first phylogenetic analysis (9), Xun et al. have shown that in contrast to PcGSTO3, PcGSTO1 is active
(17) have recently proposed from a study of the bacterial against S-glutathionyl-p-hydroquinone. Binding of S-gluta-
homologs of PcpF that the proteins belonging to the Omega thionyl-p-hydroquinone can be modeled from the PcGSTO1
subclass I constitute a new class of GSTs named S-glutathionyl- structure, one monomer of which was observed in complex
(chloro)hydroquinone reductases.
with GSH. Glutathione adopts a conformation very close to that
The present x-ray structure of PcGSTO1 confirms that it observed in HsGSTO1-1. However, on the contrary to the lat-
should be considered as the member of a new class of GSTs. ter, GSH is more buried in PcGSTO1. It results from new inter-
Indeed, the GST canonical fold (a thioredoxin-like domain fol- actions with residues that belong to the features characteristic
lowed by a ␣-helix domain) is complemented by additional spe- of PcGSTO1 as follows: Arg56 in the N-terminal coil, Trp119 in
cific features as follows: the long N-terminal coil (77 residues) the loop ␤2-␣2, and Arg155 in the loop ␣2-␤3. The additional
that covers the active site and participates in glutathione bind- coils close the cleft usually present between the Trx-like
ing, as does the long loop (about 30 residues) between the domain and the ␣-helix domain, near the loop ␤1-␣1 in which is
strand ␤2 and the ␣-helix ␣2, and finally the C-terminal coil (20 the catalytic residue Cys86. Furthermore, the side chains of
residues after the ninth ␣-helix) that allows an original Tyr223 and Tyr326 here shut up the pocket present near the
dimerization mode where the monomers interact via their catalytic cysteine in HsGSTO1-1. Even if conformational adap-
␣-helix domain (also observed for a putative GST from C. glu- tation occurs upon substrate binding, it would hardly concern
tamicum.4 All these features clearly contrast with the these residues because they belong to ␣-helices. Hence, a sub-
HsGSTO1-1 structure (10).
strate-binding site different from the putative pocket in
As shown for members of the Omega class, the main charac- HsGSTO1-1 has to be considered for the hydroquinone adduct.
teristic of GHRs is the presence of a conserved cysteine in their It could involve Trp88, Tyr223, and Phe227. All the PcGSTO1
catalytic site. From a biochemical point of view, as shown pre- residues involved either in the G-site or the putative H-site are
viously (10) and in this study, GHRs and GSTOs share the abil- conserved in the bacterial homologs (ScPcpF and EcYqjG, see
ity to reduce various substrates such as HED and DHA using Fig. 1), although they are different or absent in other GSTs (see
glutathione. Concerning PcGSTO1 and PcGSTO3, we propose supplemental Fig. 1). Once again, comparison of the PcGSTO1
from fluorescence experiments (for PcGSTO3) and free thiol and HsGSTO1-1 structures in the light of their substrate-bind-
and mass measurements that the catalytic cysteine is involved ing site emphasizes the need to consider GHRs as having a new
in the deglutathionylation activity leading to the formation of a GST structure, different from the Omega class.
disulfide bridge with the removed glutathione from their sub-
In agreement with our previous phylogenetic analysis (9),
strate. This hypothesis is in accordance with experiments per- the biochemical and structural data presented in this study
formed using thiodione or PAP-SG as substrates, because the and the recent proposition of Xun et al. (17), we propose to
appearance of the deglutathionylated products is observed in change the name of PcGSTO1 in PcGHR1 with regard to the
the absence of added glutathione, a glutathione-adduct being enzymatic function and to define a new structural class of
detected in these conditions on the resulting proteins. Further- GSTs named GST xi, characterized by their canonical GST
more, these activities require the presence of the catalytic cys- fold complemented by specific features and their new
teine. This common mechanism has been described at least for dimerization mode. Thus, this paper describes this newly
monothiol glutaredoxin (23), human GSTOs (10), and bacterial defined S-glutathionyl-(chloro)hydroquinone reductase
GHRs (16). In PcGSTO3, as shown previously for HsGSTO1-1, from P. chrysosporium as a member of the new structural
a change of the catalytic cysteine to a serine allows reversal of class xi of GSTs, which would also contain the structure of
the activity of the protein allowing the mutated protein to cat- the putative GST from C. glutamicum.
alyze glutathione transfer, and it also confirms the presence of a
As for PcpF (16), PcGHR1 in P. chrysosporium could be
disulfide bridge between Cys-37 and glutathione in PcGSTO3 involved in the catabolism of pentachlorophenol. In this fun-
WT. For PcGSTO1, biochemical data are also in accordance gus, the reductive dehalogenation of pentachlorophenol re-
with the formation of a disulfide bridge between the catalytic quires indeed at least two enzymes, a glutathione transferase for
Cys-86 and glutathione. Similar results have been obtained for glutathionylation and an enzyme called glutathione conjugate
the bacterial ortholog PcpF (16). However, this disulfide bridge reductase for deglutathionylation of the resulting S-glutathio-
is not present in the crystal structure of PcGSTO1. X-rays could nyl-p-hydroquinones (53–55). From the biochemical data pre-
have disrupted the bond, but this should have taken place very sented in this study, we postulated that PcGHR1 or the second
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GSTs of P. chrysosporium
7595–7600
isoform PcGHR2 (formerly named PcGSTO2 (9)) is responsi-
ble for this latter reaction. The wide presence of GHRs in bac-
teria, fungi, and plants suggests an additional physiological role
beyond the degradation of the recently introduced pentachlo-
rophenol. Concerning the white rot fungi, their main physio-
logical feature is their ability to mineralize wood components
and in particular lignin. It has been suggested recently that
chlorination of lignin is a phenomenon occurring through, at
least in part, the activity of chloroperoxidase (56, 57). As a
result, soils and decayed plant litter contain significant quanti-
ties of chlorinated aromatic polymers, which could lead after
oxidation via various oxidative systems to the formation of
chlorinated quinones. GHRs could play a central role in the
intracellular detoxication pathways of such molecules. On the
other hand, the significance of the Omega class extension in
saprotrophic fungi remains unclear, and the function of the
different GSTO isoforms in white rot fungi remains to be
elucidated.
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