Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis

Article abstract of DOI:10.1016/j.bbapap.2016.06.017

The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH?8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (K_D 81.2?±?9.2?mM) prior to the irreversible reaction, with maximum rate constants of 0.18?±?0.01?min^??1. Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged K_m value for GSH but showed reduced affinity for the xenobiotic substrates, higher k_cat and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed.

Full text of DOI:10.1016/j.bbapap.2016.06.017

Biochimica et Biophysica Acta 1864 (2016) 1315–1321
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
Delineation of the structural and functional role of Arg111 in GSTU4-4
from Glycine max by chemical modification and
site-directed mutagenesis
Nikolaos E. Labrou a, , Magdy Mohamed Muharram b,c, Maged Saad Abdelkader b,d
⁎
a
Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, KSA, Saudi Arabia
Department of Microbiology, College of Science, Al-Azhar University, Nasr City, 11884 Cairo, Egypt
Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria 21215, Egypt
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 April 2016
Received in revised form 20 June 2016
Accepted 29 June 2016
Available online 01 July 2016
The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical
modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inacti-
vates the enzyme in borate buffer at pH 8.0, with pseudo-first-order saturation kinetics. The rate of inactivation
exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of
reagent to the enzyme (KD 81.2 9.2 mM) prior to the irreversible reaction, with maximum rate constants of
0.18 0.01 min−1. Protection from inactivation was afforded by substrate analogues demonstrating the speci-
ficity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed
by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in sub-
strate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged Km
value for GSH but showed reduced affinity for the xenobiotic substrates, higher kcat and specific activities towards
aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the
specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xe-
nobiotic substrate specificity were discussed.
Keywords:
Chemical modification
Glutathione transferase
Molecular modelling
Site-directed mutagenesis
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
chemical carcinogens, environmental pollutants, leading to their detox-
ification [2,3,12–15]. Therefore, there, has been a particular interest in
Glutathione transferases (GSTs, EC 2.5.1.18) comprise a large family
of ubiquitous detoxifying enzymes that catalyze a wide variety of conju-
gations of glutathione (GSH) to hydrophobic electrophile compounds
[1–4]. GSTs are widespread in nature, being found in mammals, bacte-
ria, yeast, plants, insects and other sources [1–4]. GSTs form a complex
enzyme superfamily that has been subdivided into a number of classes
according to amino acid/nucleotide sequence [5,6]. GSTs from tau
(GSTU) and phi (GSTF) classes are the most abundant isoenzymes in
plants [1,3,4].
plant GSTs with regard to herbicide selectivity and environmental safe-
ty. Different classes of herbicides such as triazines, thiocarbamates,
chloroacetanilides, diphenylethers, aryloxyphenoxypropionates, etc,
can be detoxified by GSTs [7–11]. It is widely assumed that the catalytic
promiscuity of GSTs and wide substrate specificity are the results of
their structural plasticity and flexibility [1–3].
During the last decade, delineation of important structural charac-
teristics have laid the groundwork for experiments involving both ratio-
nal and random redesign of these enzymes, to create new forms of GSTs
with engineered specificities [8,12–18]. GSTs can, be considered as an
adaptable platform for engineering new catalytic activities, based on
the use of natural and catalytic diversity, particularly with respect to
the detoxification of synthetic compounds [8,12–17].
GSTs exhibit wide substrate specificity and catalyze the conjugation
of GSH with a broad range of electrophilic substrates including xenobi-
otics and endogenous electrophilic compounds such as pesticides,
Chemical modification is a useful tool for the identification and prob-
ing of specific, catalytic and regulatory sites in purified enzymes and
proteins [19]. Chemical modification experiments complement the re-
sults from crystallography and provide structural information of pro-
teins and enzymes in free solution. This approach has been widely
used to characterize several GST isoenzymes [20–21].
Abbreviations: BTD, 2,3-butanedione; CDNB, 1-chloro-2,4-dinitrobenzene; CuOOH,
cumene hydroperoxide; GSH, glutathione; GST, glutathione transferase; GSTUs, tau class
GSTs; Nb-GSH, S-nitrobenzyl-glutathione.
⁎
Corresponding author.
E-mail address: lambrou@aua.gr (N.E. Labrou).
http://dx.doi.org/10.1016/j.bbapap.2016.06.017
1570-9639/© 2016 Elsevier B.V. All rights reserved.

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N.E. Labrou et al. / Biochimica et Biophysica Acta 1864 (2016) 1315–1321
BTD [23] reacts with high rate and specificity with Arg111. The mutant
Arg111Ala enzyme exhibited unchanged Km values for GSH but showed
reduced affinity for the xenobiotic substrate and altered specificity to-
wards a range of electrophile substrates. The results may provide the
basis for the rational design of specially engineered forms of GmGSTU4-
4 with potential application in biotechnology.
2. Materials
GSH, 1-chloro-2,4-dinitrobenzene (CDNB), 2,3-butanedione (BTD),
and all other reagents and analytical grade chemicals were obtained
from Sigma-Aldrich Co (USA). Molecular biology reagents, enzymes
and kits were obtained from Invitrogen (USA).
3. Methods
3.1. Cloning, expression and purification of GmGSTU4-4
Cloning, expression and purification of GmGSTU4-4 were carried out
as described by Axarli et al. (2009) [18].
3.2. Assay of enzyme activity and protein and kinetic analysis
GST assays were performed by monitoring the formation of the con-
jugate between CDNB and GSH at 340 nm (ε = 9.6 mM−1·cm−1) ac-
cording to a published method [24]. Observed reaction velocities were
corrected for spontaneous reaction rates when necessary. Enzyme-
dependent catalysis was determined by subtracting observed rates for
conjugate formation in the absence of GmGSTU4-4 from observed
rates in the presence of GmGSTU4-4. Kinetic constants were calculated
from the initial velocities of enzyme-dependent conjugate formation.
Michaelis-Menten equation was fitted to the experimental data using
the computer program GraFit (Erithacus Software Ltd., Horley, U.K.).
All initial velocities were determined in triplicate in buffers equilibrated
at constant temperature. One unit of enzyme is defined as the amount of
enzyme that produces 1.0 μmol of product per min.
3.3. Site-directed mutagenesis
Site-directed mutagenesis was performed according to Axarli et al.,
[17]. The pairs of oligonucleotide primers used in the PCR reactions
were as follows for the Arg111Ala, 5′-GATCTTGGAGCGAAGATTTGGA
CA-3′ and 5′-TGTCCAAATCTTCGCTCCAAGATC-3′. The mutation was
verified by DNA sequencing. The mutant enzyme was expressed and
purified as described for the wild-type GmGSTU4-4 enzyme according
to [18].
Fig. 1. A. Inactivation of GmGSTU4-4 by BTD pH 8.0 and 25 °C. Enzyme (0.1 units) was
incubated in the absence (∇) of BTD or in its presence at concentrations of 10 mM (○);
20 mM (Δ); 30 mM (●); 50 mM (□); 70 mM (▲); 200 mM (■). At the times indicated,
aliquots were withdrawn and assayed for enzymatic activity. B. The effect of BTD
concentration on the observed rate of inactivation (kobs) of GmGSTU4-4 by BTD. The
concentration of BTD was in the range 10–200 mM. C. Inactivation of GmGSTU4-4 by BTD
(70 mM, ●) in the presence of 1 mM S-methyl-GSH (○) and 1 mM (□) S-nitrobenzyl-GSH.
3.4. Viscosity dependence of kinetic parameters
The effect of viscosity on kcat was studied in 0.1 M potassium phos-
phate buffer, pH 6.5, containing variable glycerol concentrations accord-
ing to [13,17,18,25]. Viscosity values (η) were calculated as described in
Wolf et al. 1985 [26]. Glycerol does not have any inhibitory effect on
GmGSTU4-4 catalysis [17,18].
In Glycine max, 42 GST isoenzymes have been identified so far [22].
Several of them have been characterized in detail [12,13,17,18]. The iso-
enzyme GmGSTU4-4 has been the major focus of interest as a model for
herbicide detoxification [9,10,11,17,18]. GmGSTU4-4 known to be the
most abundant Glycine max GST and is a homodimer protein of 214
amino acids [17,18]. GmGSTU4-4 has been explored for the develop-
ment of transgenic plants with increased resistance to abiotic stress [9,
10,11]. Therefore, detailed characterization of this enzyme is of great
importance.
3.5. Enzyme inactivation studies by BTD
Inactivation of GmGSTU4-4 or mutant Arg111Ala was performed in
1 mL of incubation mixture containing borate buffer at pH 8.0
(100 mM), BTD (10–200.0 mM) and enzyme, (0.1 units). The rate of in-
activation was followed by periodically removing samples (5–20 μL) for
assay of enzymatic activity [20,21]. Inactivation studies of GmGSTU4-4
by BTD in the presence of S-nitrobenzyl-GSH were performed in 1 mL
of incubation mixture containing borate buffer at pH 8.0 (100 mM),
BTD (70.0 mM), enzyme, (0.1 units) and S-nitrobenzyl-GSH (1 mM)
or S-methyl-GSH (1 mM). Chemical modification reaction were carried
In the present work, the structural and functional role of Arg111 in
GmGSTU4-4 was studied by chemical modification and site-directed
mutagenesis. The results showed that the arginine-specific reagent

N.E. Labrou et al. / Biochimica et Biophysica Acta 1864 (2016) 1315–1321
1317
Scheme 1. The reaction of BTD with an arginine in proteins. Borate can stabilize the adduct that is formed by arginine with BTD.
out in the dark to avoid possible photoactivation of BTD, which could
enhance nonspecific reactions with groups other than arginine [27].
where kobs is the observed rate of enzyme inactivation for a given con-
centration of BTD, k3 is the maximal rate of inactivation (min−1), and
KD is the apparent dissociation constant of the E:BTD complex. From
the data shown in Fig. 1B, a KD value of 81.2 9.2 mM and an apparent
maximal rate constant k3 of 0.18 0.01 min-1 were determined. These
results are in agreement with the view that BTD forms a reversible com-
plex GST:BTD and that formation of covalent complex is rate limiting
[21,21,31–33].
The inactivation of GmGSTU4-4 is irreversible and almost complete
restoration of activity was obtained when a sample of BTD-inactivated
enzyme was freed of BTD and borate ions using extensive dialysis or
after gel-filtration chromatography on Sephadex G-25 column. For ex-
ample, inactivated GmGSTU4-4 (b5% remaining activity) after extensive
dialysis (24 h) against 20 mM potassium phosphate buffer pH 7, re-
stores its activity (~96% reactivation). The same behaviour (~97%
reactivation) was observed when the enzyme was subjected to gel-
filtration chromatography on Sephadex G-25 column using as elution
buffer 20 mM potassium phosphate buffer pH 7. These findings confirm
that only arginyl groups are modified in GmGSTU4-4 by BTD [34,35].
The modification of arginine residues by dicarbonyl reagents (e.g.
phenylglyoxal, p-hydroxyphenylglyoxal, BTD, 1,2-cyclohexanedione,
methylglyoxal) is achieved by the reaction of the dicarbonyl group to
form cyclic adducts [23,29,30]. Studies have shown that phenylglyoxal
and 1,2-cyclohexanedione can have significant side reactions with
amino groups in the absence of borate buffers [34]. The reactions of
BTD with lysine and histidine residues are minimal especially in borate
3.6. Determination of enzyme total arginine residues
Determination of enzyme total arginine residues in native GmGSTU4-
4 as well as in the BTD-modified enzyme was carried out according to
[28]. Control incubation in the absence of GmGSTU4-4 was taken to cor-
rect the above determinations.
3.7. Biocomputing analysis
Biocomputing analysis was carried out as described in [12]. The pro-
grams PyMOL (DeLano Scientific) and UCSF Chimera were used for the
analysis of structural models.
4. Results and discussion
4.1. Kinetics of reaction of BTD with GmGSTU4-4
The modification of arginine residues in proteins is typically based
on the reaction of vicinal dicarbonyl compounds to form cyclic adducts
[19,29–30]. When GmGSTU4-4 was incubated with BTD, at pH 8.0 and
25 °C, the enzyme was progressively inactivated (Fig. 1A), whereas in
the absence of BTD, virtually no change in activity was observed when
the enzyme was incubated under identical conditions. The observed
rate of inactivation was dependent upon BTD concentration as shown
in Fig. 1 (A & B). This indicated that the reaction obeyed pseudo-first
order saturation kinetics and was consistent with a Kitz & Wilson
model [31] for reversible binding of reagent (BTD), prior to covalent
modification according to equation [20,21,31-33]:
KD
k3
E þ BTD ⇄ E : BTD → E‐BTD
where E represents the free enzyme; E:BTD is the reversible complex
and E-BTD is the covalent product. The steady-state rate equation for
the interaction is [31–33]:
kobs ¼ k3 ½BTDꢀ=ðKD þ ½BTDꢀÞ
Table 1
Total arginine determination in the unmodified and BTD-modified GmGSTU4-4. The anal-
ysis was performed in three separate experiments. According to enzyme's primary struc-
ture, GmGSTU4-4 has nine arginine residues.
Unmodified enzyme
(mol of Arg residue/mol
enzyme subunit)
Modified enzyme
(mol of Arg residue/mol
enzyme subunit)
Difference
Fig. 2. Structural representation depicting Arg111 and other important residues of
GmGSTU4-4. The bound ligand (S-nitrobenzyl-GSH, Nb-GSH) and Arg111 are colored
according to atom type and represented as thick sticks. Other active site side chains are
represented as thin sticks. S-nitrobenzyl-GSH and Arg111 are labelled.
9.14
8.82
9.33
8.24
7.89
8.42
0.90
0.93
0.91

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aspartic and glutamic acid residues and form salt bridges which reduces
their reactivity [34,35].
To provide further experimental evidence and establish the involve-
ment or not of Arg111 in the reaction with BTD, site-directed mutagen-
esis of Arg111 was employed. Arg111 was mutated to Ala and the
mutant enzyme was expressed, purified and subjected to chemical
modification by BTD, under exactly the same conditions to those used
for the wild-type enzyme. The results showed that the Arg111Ala mu-
tant enzyme is resistant to inactivation by BTD and retains all of its ac-
tivity (Fig. 3). Comparison of the far UV difference spectra of native
and mutated enzyme (data not shown) indicated the absence of any
structural perturbation caused by the mutation. This rules out the possi-
bility that the resistance to inactivation of the Arg111Ala-mutated en-
zyme is due to conformational changes in the structure of the enzyme.
4.3. The role of Arg111 in substrate binding and catalysis
Fig. 3. Time course of inactivation of the wild-type GmGSTU4-4 and mutant Arg111Ala by
BTD. Wild type (●) and mutant Arg111Ala (□) were incubated in the presence of 100 mM
BTD at pH 8.0 and 25 °C. At the times indicated, aliquots were withdrawn and assayed for
enzymatic activity.
Aminoacid sequence alignments of tau class GSTs [18] suggest that
Arg111 is not conserved among other tau class GSTs, indicating a specific
role of this residue in GmGSTU4-4. Kinetic analysis of Arg111Ala mutant
enzyme using the model substrates CDNB and cumene hydroperoxide
was carried out and the results are listed in Tables 2 & 3. The results
showed that the mutation does not appreciably alter the affinity of the
enzyme for GSH. On the other hand, the mutant enzyme displays in-
creased Km values for CDNB and cumene hydroperoxide although its
catatalytic efficiency (kcat/Km) towards CDNB and cumene hydroperoxide
remained essentially unaltered. Interestingly, more profound effect of the
mutation was observed on kcat value. The mutant enzyme exhibits about
four times higher kcat value (Tables 2 & 3), compared to the wild type
enzyme, suggesting the involvement of this amino acid to the catalytic
mechanism.
GmGSTU4-4 catalyzes a broad range of reactions [18] and therefore
to evaluate the contribution of the mutation on the enzyme's activity to-
wards different electrophile substrates, a broad range of substrates was
examined and the results are listed in Table 4. The wild-type and mutant
enzymes were assayed for activities as a glutathione transferase and as a
glutathione peroxidase. The results indicate that the contribution of
Arg111 depends on the electrophile substrate. In particular, with all ar-
omatic substrates tested (p-nitrobenzyl chloride, atrazine, alachlor,
ethacrynic acid, phenethyl isothiocyanate) the enzyme displays in-
crease specific activity, compared to the wild-type enzyme. On the
other hand using smaller aliphatic substrates, such as tert-butyl hydro-
peroxide, allyl isothiocyanate and trans-2-nonenal, the enzyme exhibits
lower or unchanged specific activity, compared to the wild-type en-
zyme. This observation suggests a specific role of Arg111 in binding
large aromatic substrates.
buffers [35]. As shown in Scheme 1, borate anions can form complex
with the diol group and therefore stabilize the initial adduct and there-
fore prevent the regeneration of arginine [35]. The specificity of the BTD
interaction with GmGSTU4-4 is evidenced from the stoichiometry of in-
corporation. In order to identify the amino acid residue modified, amino
acid analysis was employed. Total arginine determination was carried
out for the modified as well as for the unmodified enzyme. The results
(Table 1) from a total arginine determination indicated that the BTD-
modified enzyme shows loss of 0.91
0.17 mol of Arg/mol enzyme
subunit, which is close to unity, suggesting that BTD reacts with a single
Arg residue in each enzyme subunit.
The ability of specific ligands (e.g. substrates and inhibitors) to pre-
vent enzyme inactivation by an irreversible inhibitor is the usual criteri-
on used in arguing for active site-directed modification [20,21,32,33].
The inactivation of GmGSTU4-4 was reduced significantly by the pres-
ence of S-nitrobenzyl-GSH as illustrated in Fig. 1C. In the presence of
S-methyl-GSH or S-nitrobenzyl-GSH the enzyme showed about 30%
and 65%, lower inactivation rates, respectively, compared to that in
the absence of the inhibitors. Considering the structures of S-
nitrobenzyl-GSH and S-methyl-GSH it is reasonable to assume that the
target arginine residue is located at the H-site and protected more effi-
ciently by large xenobiotic compounds (e.g. nitrobenzyl group) com-
pared to the smaller structures (e.g. methyl group).
4.2. Identification of the Arg residue modified by BTD
In order to gain a deeper insight into the role of Arg111 in the catalytic
mechanism of GmGSTU4-4, analysis of the available crystal structures of
the enzyme [17,18] in complex with S-nitrobenzyl-GSH (reaction product
analogue; PDB ID: 4VO4) and GSH (substrate; PDB ID: 4TOP) was under-
taken. A striking difference between the structure of the enzyme·S-
nitrobenzyl-GSH complex and the structure enzyme·GSH is the move-
ment of the side chain of Arg111. In the enzyme·S-nitrobenzyl-GSH
structure, the guanidine group of this residue is in hydrogen-bonding
From the analysis of the crystal structure of the enzyme [17,18] it is
evident that one arginine residue (Arg111) is located on the topmost re-
gion of α-helix H4 and projects into the large H-site (Fig. 2). Its side
chain is accessible to the solvent and therefore is accessible for covalent
modification by BTD. Although arginines are almost always found on
protein surfaces in large numbers, their reactions with dicarbonyl com-
pounds seem to be low. This is due to their ability to interact with
Table 2
Steady-state kinetic parameters of GmGSTU4-4 and Arg111Ala mutant enzyme for the CDNB/GSH reaction.
Enzyme
Km, (mM)
(GSH)
Km, (mM)
(CDNB)
kcat
k
cat/KCmDNB
k
cat/KGmSH
(s−1
)
(s−1 mM−1
)
(s−1 mM−1
)
Wild-typea
Arg111Ala
0.159 0.019
0.137 0.073
0.15 80.0316
0.40 0.039
2.48 0.31
8.04 0.18
15.7
20.1
15.6
58.7
a
The kinetic parameters for the wild-type enzyme were taken from reference [18] and included for comparison.

N.E. Labrou et al. / Biochimica et Biophysica Acta 1864 (2016) 1315–1321
1319
Table 3
Steady-state kinetic parameters of GmGSTU4-4 and Arg111Ala mutant enzyme for the cumene hydroperoxide/GSH reaction.
Enzyme
Km, (mM)
(GSH)
Km, (mM)
(CuOOH)
kcat
kcat/KCmuOOH
(s−1 mM−1
kcat/KGmSH
(s−1
)
)
(s−1 mM−1
)
Wild-typea
Arg111Ala
0.036 0.008
0.048 0.01
0.45 0.084
0.82 0.07
0.24 0.01
0.62 0.02
0.53
0.76
6.7
12.9
a
The kinetic parameters for the wild-type enzyme were taken from reference [18] and included for comparison.
distance (2.7 Å) with the hydroxyl group of Tyr107, whereas in the GSH-
complexed enzyme, the distance is about 5 Å (Fig. 4A). The hydroxyl
group of Tyr107 makes an on-face hydrogen bond with the π-electron
cloud of the benzyl group of the bound S-nitrobenzyl-GSH. This interac-
tion may stabilize aromatic substrates at their productive orientation.
The loss of side-chain of Arg in the Arg111Ala mutant enzyme may influ-
ence the position of Tyr107 that may lead to higher Km for aromatic sub-
strates (CDNB, cumene hydroperoxide, Tables 2 & 3). Fig. 4 shows the in
silico structural study of the wild type (left column) and the Arg111Ala
mutant enzymes (right column). In particular, Fig 4C, left, shows that
there is no direct interaction between Arg111 and GSH. The interaction
energy of the ligand in the wild-type enzyme (PDB ID: 4VO4) was calcu-
lated 101.175 kcal/mol2. In the mutant enzyme the free interaction ener-
gy of the ligand was found to be the same. This interaction energy-based
comparison confirms the findings from the kinetic analysis (Tables 2 and
3), which showed that the Km for GSH remained unchanged in the mutant
enzyme. However, looking closer into the dynamics of the protein and
the route that the ligand has to follow in order to occupy its position
in the active site we can suggest that the bulkier side-chain of Arg111
residue, compared to that of Ala residue in the mutant enzyme controls
the entrance of the active site. However, as soon as the ligand positions
itself in the active site of the wild-type enzyme, the side-chain of
Arg111 residue re-occupies its space with total energy of
−38.516 kcal/mol2. Negative interaction energies are indicative of sta-
ble molecular systems. On the contrary, upon the Arg111Ala mutation
the smaller Ala residue displayed total energy of 16.245 kcal/mol2,
which indicates that the Ala residue is unstable and the adaptation of
the position of the original side-chain of Arg residue is unfavourable.
This can be explained by Fig 4C, where the interaction between the
Ala111 residue with other neighbour residues are depicted. A new pi-
stacking interaction has been established with Trp114 (Fig 4C, right),
which is clearly pushing and locking the Ala111 residue in a single
Table 4
Substrate specificity for GmGSTU4-4 and Arg111Ala mutant enzyme. Enzyme assays were carried out under standard conditions as described in the Methods section. The specific activity
for the wild-type enzyme was taken from reference [18] and included for comparison. Results represent the means of triplicate determinations, with variation b5% in all cases.
Substrate
Structure
GmGSTU4-4
Arg111Ala mutant
Specific activity
Specific activity
(Units/mg protein)
(Units/mg protein)
(×10−1
)
(×10−1
)
p-Nitrobenzyl chloride
2.2
4.2
Atrazine
Alachlor
0.02
0.04
0.04
0.09
tert-Butyl hydroperoxide
trans-2-Nonenal
0.85
0.93
0.21
0.81
2,3-Dichloro-4-[2-methylene-butyryl]phenoxy acetic acid (ethacrynic acid)
0.53
3.4
Phenethyl isothiocyanate
Allyl isothiocyanate
1.84
0.63
2.9
0.62

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high-energy trap position. Fig. 4D shows a structural superposition of
the introduced Ala residue (in ball and stick representation) in the 3D
conformational space that Arg111 originally occupied (in 3D cloud/sur-
face representation). It is clear that the small side chain of the alanine
residue is no substitute for the bulky Arg side chain. The entrance to
the active site remains constantly open and the ligand can move freely
in and out and as a consequently, the regulation of kcat is expected to be
observed in this molecular system (Tables 2 and 3).
Recent studies on the catalytic mechanism of GmGSTU4-4 with
CDNB as co-substrate have established that the rate-limiting step in
the enzyme is not dependent on a diffusional barrier (e.g. product re-
lease) [18]. Instead, other viscosity-dependent motions or conforma-
tional changes of the protein, contribute to the rate-limiting step [18].
As illustrated in Fig. 5, the mutant Arg111Ala, exhibits different depen-
dence on viscosity, compared to the wild-type enzyme, suggesting that
the mutation affects the rate-limiting step of the catalytic reaction.
Structural comparison (Fig. 4A) between the GSH-bound (enzyme-sub-
strate complex) [17] and S-nitrobenzyl-GSH bound enzyme (enzyme-
product complex) [18] revealed that large conformational changes oc-
curs in the upper part of α-helix H4 (where Arg111 is located), in the
C-terminal region (α-helix H9) as well as in the loop the connects α-
helices H4 and H5. These structural transitions as well as dynamic issues
may contribute to the observed differences in the rate-limiting step of
the mutant enzyme.
4.4. Biological relevance
Dicarbonyl compounds such as BTD, methylglyoxal are cytotoxic by-
products that are formed endogenously via different enzymatic and
non-enzymatic reactions [36–40]. It is well established that dicarbonyl
compounds initiate stress-induced signalling cascade via reactive oxy-
gen species, resulting in the modifications of proteins involved in vari-
ous signal transduction pathways, that eventually promotes in cell
death or growth arrest [38]. The concentration of dicarbonyl com-
pounds is varies in different plant species and increases in response to
abiotic stress such as salinity, drought, and cold stress conditions [36].
We have recently showed that GmGSTU4 gene expression displays a
specific induction patterns. In particular, GmGSTU4 gene is over
expressed (approximately 153-fold) at RNA level under salinity stress
caused by NaCl [10]. Considering the specificity of chemical modifica-
tion of Arg111 as well as the highly specific stress-inducible nature of
this enzyme under conditions which promote the elevation of
dicarbonyl compounds levels (e.g. salt stress) [36], it is conceivable to
assume an in vivo relevant functional role of GmGSTU4-4/dicarbonyl in-
teraction and enzyme inactivation.
5. Conclusion
In the present work we presented a combination of protein chemis-
try and protein engineering studies to characterize an important amino
acid residue in GmGSTU4-4. The inactivation of GmGSTU4-4 by BTD ex-
hibited pseudo-first order saturation kinetics and about 1 mol of Arg/
mol of enzyme subunit is modified upon complete inactivation. In addi-
tion, the active-site inhibitors S-nitrobenzyl-GSH and S-methyl-GSH
provided protection against inactivation and the Arg111Ala mutant en-
zyme is resistant to inactivation. All these findings point to the conclu-
sion that Arg111 is the target for modification by BTD. Kinetic analysis
established that Arg111 contributes to xenobiotic substrate binding
and catalysis by influencing the Km and kcat values. These findings
Fig. 4. A. Structural rearrangements in GmGSTU4-4. Superposition of GmGSTU4-4 struc-
ture in complex with S-nitrobenzyl-GSH (Nb-GSH, light brown) and GSH (light blue).
The bound ligands (S-nitrobenzyl-GSH, and GSH) are colored according to atom type.
Arg111 and Tyr107 are represented as sticks. S-nitrobenzyl-GSH, Arg111 and Tyr107
and the α-Helices H4 and H9 are labelled. (B). Molecular modelling study of the wild-type
enzyme (left column) and the Arg111Ala mutant enzyme (right column). A zoomed-in
view of the area in the proximity of position 111 and the ligand. (C). A schematic 2D inter-
action plot for both the wild-type enzyme and the mutant Arg111Ala residues. (D) A struc-
tural superposition of the wild-type and mutant Arg111Ala 3D structures. The Ala residue
is depicted in ball and stick representation, the GSH ligand is colored green and the original
3D conformational space of the Arg111 residue is defined by a 3D electrostatic cloud/sur-
face representation. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

N.E. Labrou et al. / Biochimica et Biophysica Acta 1864 (2016) 1315–1321
1321
[11] C. Kissoudis, C. Kalloniati, E. Flemetakis, P. Madesis, N.E. Labrou, A. Tsaftaris, I.
Nianiou-Obeidat, Maintenance of metabolic homeostasis and induction of
cytoprotectants and secondary metabolites in alachlor treated GmGSTU4 overex-
pressing tobacco plants, as resolved by metabolomics, Plant Biotechnol. Rep. 9
(2015) 287–296.
[12] I. Axarli, A.W. Muleta, D. Vlachakis, S. Kossida, G. Kotzia, A. Maltezos, P. Dhavala, A.C.
Papageorgiou, N.E. Labrou, Directed evolution of Tau class glutathione transferases
reveals a site that regulates catalytic efficiency and masks co-operativity, Biochem.
J. 473 (2016) 559–570.
[13] K. Skopelitou, A.W. Muleta, A.C. Papageorgiou, E. Chronopoulou, N.E. Labrou, Cata-
lytic features and crystal structure of a tau class glutathione transferase from Glycine
max specifically upregulated in response to soybean mosaic virus infections,
Biochim. Biophys. Acta 1854 (2015) 166–177.
[14] K. Skopelitou, P. Dhavala, A.C. Papageorgiou, N.E. Labrou, A glutathione transferase
from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily,
PLoS One 7 (2012), e34263.
[15] W. Zhang, D.F. Dourado, B. Mannervik, Evolution of the active site of human gluta-
thione transferase A2-2 for enhanced activity with dietary isothiocyanates, Biochim.
Biophys. Acta 1850 (2015) 742–749.
[16] A. Runarsdottir, B. Mannervik, A novel quasi-species of glutathione transferase with
high activity towards naturally occurring isothiocyanates evolves from promiscuous
low-activity variants, J. Mol. Biol. 401 (2010) 451–464.
[17] I. Axarli, P. Dhavala, A.C. Papageorgiou, N.E. Labrou, Crystal structure of Glycine max
glutathione transferase in complex with glutathione: investigation of the mecha-
nism operating by the Tau class glutathione transferases, Biochem. J. 422 (2009)
247–256.
[18] I. Axarli, P. Dhavala, A.C. Papageorgiou, N.E. Labrou, Crystallographic and functional
characterization of the fluorodifen-inducible glutathione transferase from Glycine
max reveals an active site topography suited for diphenylether herbicides and a
novel L-site, J. Mol. Biol. 385 (2009) 984–1002.
Fig. 5. The effect of viscosity on kcat. The effect of viscosity on kcat for the CDNB/GSH
reaction catalyzed by GmGSTU4-4 and its Arg111Ala mutant. Plot of the reciprocal of the
relative turnover number (kocat/kcat) as a function of relative viscosity (η/ηo) with
glycerol as cosolvent for the wild-type, (○); and for the Arg111Ala (●) mutant enzyme.
Lines were calculated by least-squares regression analysis.
[19] V.L. Mendoza, R.W. Vachet, Probing protein structure by amino acid-specific cova-
lent labeling and mass spectrometry, Mass Spectrom. Rev. 28 (2009) 785–815.
[20] I.A. Axarl, D.J. Rigden, N.E. Labrou, Characterization of the ligandin site of maize glu-
tathione S-transferase I, Biochem. J. 382 (2004) 885–893.
[21] G.A. Kotzia, N.E. Labrou, S-(2,3-dichlorotriazinyl)glutathione, A new affinity label for
probing the structure and function of glutathione transferases, Eur J Biochem 271
(2004) 3503–3511.
have relevance for future efforts aiming to redesign xenobiotic substrate
specificity. In addition, the association of dicarbonyl compounds with
plant stress response may indicate an in planta role of GmGSTU4-4/
dicarbonyl interaction.
[22] B. McGonigle, S.J. Keeler, S.M. Lau, M.K. Koeppe, D.P.A. O'Keefe, Genomics approach
to the comprehensive analysis of the glutathione S-transferase gene family in soy-
bean and maize, Plant Physiol. 124 (2000) 1105–1120.
[23] G. Chen, X. Chen, Arginine residues in the active site of human phenol
sulfotransferase (SULT1A1), J. Biol. Chem. 278 (2003) 36358–36364.
[24] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferases. The first enzymatic
step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139.
[25] W.W. Johnson, S. Liu, X. Ji, G.L. Gilliland, R.N. Armstrong, Tyrosine 115 participates
both in chemical and physical steps of the catalytic mechanism of a glutathione S-
transferase, J. Biol. Chem. 268 (1993) 11508–11511.
[26] A.V. Wolf, M.G. Brown, P.G. Prentiss, R.C. Weast, M.J. Astle, W.H. Beyer (Eds.), Hand-
book of Chemistry and Physics, CRC Press, Inc., Boca Raton, F.L. 1985, pp. D-219–D-
269.
[27] H. Fliss, T. Viswanatha, 2,3-Butanedione as a photosensitizing agent: application to
alpha-amino acids and alpha-chymotrypsin, Can. J. Biochem. 57 (1979) 1267–1272.
[28] R.B. Yamasaki, D.A. Shimer, R.E. Feeney, Colorimetric determination of arginine res-
idues in proteins by p-nitrophenylglyoxal, Anal. Biochem. 111 (1981) 220–226.
[29] B.R. Epperly, E.E. Dekker, Inactivation of Escherichia coli L-threonine dehydrogenase
by 2,3-butanedione. Evidence for a catalytically essential arginine residue, J. Biol.
Chem. 264 (1989) 18296–18301.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgements
This project was supported by the Deanship of Scientific Research at
Prince Sattam Bin Abdulaziz University under the research project
2015/03/4385. We acknowledge the assistance of Dr. D. Vlachakis in
molecular modelling.
References
[1] N.E. Labrou, A.C. Papageorgiou, O. Pavli, E. Flemetakis, Plant GSTome: structure and
functional role in xenome network and plant stress response, Curr. Opin. Biotechnol.
32C (2015) 186–194.
[30] C.J. Vlahos, M.A. Ghalambor, E.E. Dekker, Evidence for an essential arginine residue
in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. Modification
with 1,2-cyclohexanedione, J. Biol. Chem. 260 (1985) 5480–5485.
[31] R. Kitz, I.B. Wilson, Esters of methanesulfonic acid as irreversible inhibitors of acetyl-
cholinesterase, J. Biol. Chem. 237 (1962) 3245–3249.
[2] B. Mannervik, Five decades with glutathione and the GSTome, J. Biol. Chem. 287
(2012) 6072–6083.
[3] I. Cummins, D. Dixon, Multiple roles for plant glutathione transferases in xenobiotic
detoxification, Drug Metab. Rev. 43 (2011) 266–280.
[4] I. Cummins, D.J. Wortley, F. Sabbadin, Z. He, C.R. Coxon, H.E. Straker, J.D. Sellars, K.
Knight, L. Edwards, D. Hughes, et al., Key role for a glutathione transferase in
multiple-herbicide resistance in grass weeds, Proc. Natl. Acad. Sci. U. S. A. 110 (2013)
5812–5817.
[32] R.F. Colman, Chemical arrows for enzymatic targets, FASEB J. 11 (1997) 217–226.
[33] N.E. Pettigrew, E.J. Brush, R.F. Colman, 3-Methyleneoxindole: an affinity label of glu-
tathione S-transferase pi which targets tryptophan 38, Biochemistry 40 (2001)
7549–7558.
[5] Y.-J. Liu, X.-M. Han, L.-L. Ren, H.-L. Yang, Q.-Y. Zeng, Functional divergence of the gluta-
thione S-transferase supergene family in Physcomitrella patens reveals complex pat-
terns of large gene family evolution in land plants, Plant Physiol. 161 (2013) 773–786.
[6] P.-A. Lallement, E. Meux, J.M. Gualberto, P. Prosper, C. Didierjean, F. Saul, A. Haouz,
N. Rouhier, A. Hecker, Structural and enzymatic insights into Lambda glutathione
transferases from Populus trichocarpa, monomeric enzymes constituting an early di-
vergent class specific to terrestrial plants, Biochem. J. 462 (2014) 39–52.
[7] R. Sharma, A. Sahoo, R. Devendran, M. Jain, Over-expression of a rice tau class gluta-
thione S-transferase gene improves tolerance to salinity and oxidative stresses in
Arabidopsis, PLoS One 9 (2014), e92900.
[8] D.P. Dixon, A.G. McEwen, A.J. Lapthorn, R. Edwards, Forced evolution of a herbicide
detoxifying glutathione transferase, J. Biol. Chem. 278 (2003) 23930–23935.
[9] K. Benekos, C. Kissoudis, I. Nianiou-Obeidat, N.E. Labrou, P. Madesis, M. Kalamaki, A.
Makris, A. Tsaftaris, Over-expression of a specific soybean GmGSTU4 isoenzyme im-
proves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic to-
bacco plants, J. Biotechnol. 150 (2010) 195–201.
[34] L. Patthy, E.L. Smith, Reversible modification of arginine residues, J. Biol. Chem. 250
(1975) 557–564.
[35] J.F. Riordan, Arginyl residues and anion binding sites in proteins, Mol. Cell. Biochem.
26 (1979) 71–92.
[36] T.H. Phang, G. Shao, H.M. Lam, Salt tolerance in soybean, J. Integr. Plant Biol. 50
(2008) 1196–1212.
[37] S.K. Yadav, S.L. Singla-Pareek, M. Ray, M.K. Reddy, S.K. Sopory, Methylglyoxal levels
in plants under salinity stress are dependent on glyoxalase I and glutathione,
Biochem. Biophys. Res. Commun. 337 (2005) 61–67.
[38] S.K. Yadav, S.L. Singla-Pareek, S.K. Sopory, An overview on the role of methylglyoxal
and glyoxalases in plants, Drug Metabol. Drug Interact. 23 (2008) 51–68.
[39] J.M. Mathews, S.L. Watson, R.W. Snyder, J.P. Burgess, D.L. Morgan, Reaction of the
butter flavorant diacetyl (2,3-butanedione) with N-α-acetylarginine: a model for
epitope formation with pulmonary proteins in the etiology of obliterative bronchi-
olitis, J. Agric. Food Chem. 58 (2010) 12761–12768.
[40] P.R. Lozano, M. Drake, D. Benitez, K.R. Cadwallader, Instrumental and sensory char-
acterization of heat-induced odorants in aseptically packaged soy milk, J. Agric. Food
Chem. 55 (2007) 3018–3026.
[10] C. Kissoudis, C. Kalloniati, O. Pavli, E. Flemetakis, N.E. Labrou, P. Madesis, G. Skaracis,
A. Tsaftaris, I. Nianiou-Obeidat, Stress inducible GmGSTU4 shapes transgenic tobac-
co plants metabolome towards increased salinity tolerance, Acta Physiol. Plant. 37
(2015) 102.