Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase

Article abstract of DOI:10.1016/j.bbagen.2012.04.022

Background: Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its

Full text of DOI:10.1016/j.bbagen.2012.04.022

Biochimica et Biophysica Acta 1820 (2012) 1469–1474
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase
a,
b
a
a
c
Kohji Yamamoto ⁎, Kazuhiro Usuda , Yoshimitsu Kakuta , Makoto Kimura , Akifumi Higashiura ,
c
a
c
Atsushi Nakagawa , Yoichi Aso , Mamoru Suzuki
Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812‐8581, Japan
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812‐8581, Japan
Institute for Protein Research, Osaka University, Suita 565‐0871, Japan
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway
for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST
from Bombyx mori (bmGSTD) to examine its catalytic residues.
Methods: The three-dimensional structure of bmGSTD was resolved by the molecular replacement method
and refined to a resolution of 2.0 Å.
Received 9 February 2012
Received in revised form 27 April 2012
Accepted 30 April 2012
Available online 8 May 2012
Results: Structural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2
distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound gluta-
thione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-
directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glu-
tathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants
indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function.
General significance: These results provide structural insights into the catalysis of glutathione conjugation in
B. mori by bmGSTD.
Keywords:
Crystal structure
Glutathione
Glutathione transferase
Lepidoptera
Site-directed mutagenesis
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
Among the lepidopteran GSTs, we found that the Delta-class GST
of B. mori (bmGSTD) is involved in detoxification of intrinsic alde-
hydes produced by lipid peroxidation [8]. Since a number of agricul-
tural pests belong to the Lepidoptera, a better understanding of the
catalytic activity of silkworm GSTs would enhance control manage-
ment for agricultural pests. However, structural data for bmGSTD
are not available, and more information on the biochemical and struc-
tural properties of B. mori GSTs is required. In this study, we deter-
mined the three-dimensional structure of bmGSTD, and investigated
structure–function relationships in enzyme catalysis using recombi-
nant bmGSTD obtained by overexpression in Escherichia coli. Results
from site-directed mutagenesis suggested that conserved amino
acid residues in the GSH-binding region of bmGSTD are critical for
enzyme catalysis.
The glutathione transferases (GSTs) [EC 2.5.1.18], widely distrib-
uted in various organisms, are enzymes responsible for the detoxifi-
cation of exogenous and endogenous substances, by conjugation
with reduced glutathione (GSH) [1,2]. The GST enzyme superfamily
comprises various classes: Alpha, Mu, Pi, Omega, Sigma, Theta, and
Zeta in mammals [3]; and Delta, Epsilon, Omega, Sigma, Theta, and
Zeta in dipteran insects such as Anopheles gambiae and Drosophila
melanogaster [4]. Insect GSTs have attracted a great deal of interest,
as they are involved in insecticide metabolism [5–7]. Hence, we
have characterized Sigma-, Omega-, Zeta-, and Delta-class GSTs in
the silkworm, Bombyx mori, a lepidopteran model insect [8–12], and
a Sigma-class GST in the fall webworm, Hyphantria cunea [13]. Anoth-
er, previously unidentified insecticide-induced GST class from B. mori,
bmGSTu, has been recently identified and characterized [14,15].
2. Materials and methods
2.1. Crystallization procedures
Abbreviations: CBB, Coomassie Brilliant Blue R250; CDNB, 1-chloro-2,4-dinitrobenzene;
ECA, ethacrynic acid; GTX, S-hexyl glutathione; GSH, glutathione; GST, glutathione transfer-
ase; 4-HNE, 4-hydroxynonenal; IPTG, isopropyl 1-thio-β-D-galactoside; LB, Luria–Bertani
medium; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; PEG,
polyethylene glycol; ROS, reactive oxygen species; SDS‐PAGE, sodium dodecyl sulfate‐
polyacrylamide gel electrophoresis
The bmGSTD protein was purified according to the method previ-
ously reported [8], and concentrated to approximately 10 mg/ml in
20 mM Tris–HCl buffer, pH 8.0, containing 0.2 M NaCl. Crystallization
was carried out by the sitting-drop vapor diffusion method at 20 °C
using crystal screen kits (Hampton research), including PEG ION-1
and ‐2, Index, Crystal Screen-1 and ‐2, and Wizard-1 and ‐2, as reser-
voir solutions. Each drop was formed by mixing equal volumes
⁎
Corresponding author at: 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812‐8581, Japan.
Tel.: +81 92 621 4991; fax: +81 92 624 1011.
E-mail address: yamamok@agr.kyushu-u.ac.jp (K. Yamamoto).
0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2012.04.022

1470
K. Yamamoto et al. / Biochimica et Biophysica Acta 1820 (2012) 1469–1474
(
0.2:0.2 μl) of protein and reservoir solution. Crystals to be used for
[8] and the Quick-Change Site-Directed Mutagenesis Kit according
to the manufacturer's recommendations (Agilent Technologies,
Wilmington, USA). Full-length mutant cDNAs were systematically
checked by DNA sequencing. Homology alignment was performed
using ClustalW (ver. 1.83), with 10 and 0.2 as the gap creation penalty
and the gap extension, respectively.
X-ray analysis were grown at 20 °C for 1 month with 0.2 M sodium
acetate trihydrate, 0.1 M sodium cacodylate trihydrate (pH 6.5), and
3
using synchrotron radiation at beamline BL38B1 of SPring-8 under
cryogenic conditions with crystals soaked in a cryoprotectant solu-
tion containing 20% (v/v) glycerol and cooled to 100 K in a nitrogen
gas stream. The diffraction data were processed using the HKL2000
program package [16].
0% (w/v) PEG 8000. The X-ray diffraction data set was collected
2.4. Overexpression and purification of recombinant protein
Mutant bmGSTD was cloned into the expression vector pET-11b
(
Novagen) and transformed into competent E. coli Rosetta (DE3)
2
.2. Determination of crystal structure
pLysS cells (Novagen). Cells were then grown at 37 °C in Luria–
Bertani (LB) media containing 100 μg/ml ampicillin. After the cell
density reached 0.7 at OD600, isopropyl 1-thio-β-D-galactoside (IPTG)
was added to obtain a final concentration of 1 mM, required to induce
the production of recombinant protein. After further incubation for
The crystal structure of bmGSTD was determined by the molec-
ular replacement method using the program MOLREP [17] based
on a comparison with the previously reported structure of A.
gambiae Delta-class GST, agGSTd1-6 (PDB ID: 1PN9). The structure
was refined using the program REFMAC5 [18] with diffraction data
from 2.0 Å resolution for bmGSTD. A manual model fitting to the
electron density maps was performed using the program Coot
3
h, cells were harvested by centrifugation, resuspended in 20 mM
Tris–HCl, pH 8.0, 0.5 M NaCl, 4 mg/ml lysozyme, and 1 mM
phenylmethanesulfonyl fluoride, and disrupted by sonication.
Unless otherwise noted, all operations described below were
conducted at 4 °C. The supernatant was clarified by centrifugation
at 10,000×g for 15 min and subjected to ammonium sulfate
fractionation. The pellet obtained by 30%–70% salt saturation was
resuspended in a 10 mM sodium phosphate buffer, pH 8.7. After
dialysis against the same buffer, the specimen was subjected to
[19]. The stereochemical quality of the final model was assessed
by the PROCHECK [20], PISA [21], and DSSP [22] programs. The
crystallographic parameters and refinement statistics are summa-
rized in Table 1. All figures were prepared by using PyMOL
(
http://pymol.sourceforge.net). The atomic coordinates and struc-
tural factors of bmGSTD have been deposited in the Protein Data
Bank (PDB ID: 3VK9).
anion-exchange chromatography on
Healthcare) column, and eluted with a linear gradient of 0 to
.3 M NaCl. Fractions containing the enzyme were pooled, con-
a DEAE‐Sepharose (GE
0
2
.3. Site-directed mutagenesis
centrated using a centrifugal filter (Millipore), and applied to a
Superdex 200 column (GE Healthcare) equilibrated with the
same buffer containing 0.2 M NaCl. Sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS‐PAGE) was performed using
a 15% polyacrylamide slab gel containing 0.1% SDS according to
the method of Laemmli [23]. Protein bands were visualized by
staining with Coomassie Brilliant Blue R250 (CBB). Protein con-
centration was measured using a Protein Assay Kit (Bio-Rad Lab-
oratories) with bovine serum albumin as a standard.
Amino acid-substituted mutants of bmGSTD were constructed
using a previously prepared plasmid of bmGSTD wild-type enzyme
Table 1
Data collection and refinement statistics for bmGSTD.
Parameter
bmGSTD
C222
a=128.4, b=162.2, c=86.79
α=β=γ=90.00
SPring-8 BL38B1
1.000
32.9–2.00 (2.07–2.00)
311,763
60,864 (6035)
5.1 (5.1)
Space group
Unit cell parameters (Å)
1
2
.5. Measurements of enzyme activity
(°)
Beam line
Wavelength (Å)
Resolution range (Å)
Total number of observation reflections
Total number of unique reflections
Under standard assay conditions, GST activity was spectrophoto-
metrically measured using 1-chloro-2,4-dinitrobenzene (CDNB) and
GSH (γ-Glu-Cys-Gly) [14,24]. Briefly, 0.01 ml of test solution was
added to 1 ml of 50 mM sodium phosphate buffer, pH 6.5, con-
taining 0.5 mM CDNB and 5 mM GSH as substrates. Changes in ab-
sorbance (340 nm/min) were monitored at 30 °C and converted
into moles CDNB conjugated/min/mg protein using the molar ex-
tinction coefficient of the resultant 2,4-dinitrophenyl-glutathione:
Multiplicity
b
R
merge (%)
18.6 (75.9)
8.51 (1.77)
99.3 (99.7)
a
bI>/bσ (I)>
Completeness (%)
Refinement statistics
Resolution range (Å)
Number of reflections
Working set/test set
−
1
−1
ε
340 =9600 M
m
cm . Kinetic parameters (K and Vmax) were
32.9–2.00
obtained from a double-reciprocal plot of data generated by varying
the concentrations of CDNB or GSH using the program Kaleida
Graph (HULINKS Inc.).
57,790/3060
26.0/21.3
c
d
R
work (%)/Rfree (%)
Root-mean-square deviations
Bond lengths (Å)/bond angles (°)
Average B-factors (Å )/number of atoms
0.007/1.041
2
3. Results
Protein
Glycerol
20.2/6952
44.4/54
3.1. X-ray analyses of recombinant bmGSTD
Water
23.6/375
Ramachandran analysis
Preferred regions (%)
Allowed regions (%)
Outliers (%)
After bacterial expression, recombinant bmGSTD was purified
97.2
2.8
0.0
and crystallized. The crystallized protein possessed a space group
of C222 with unit cell dimensions a=128.4 Å, b=162.2 Å, and
1
a
Values in parentheses are for the highest-resolution shell.
c=86.79 Å. The structure was solved and the phases were refined;
relevant data are listed in Table 1. The final Rwork and Rfree factors
were 26.0% and 21.3% for resolutions of 32.9 Å and 2.0 Å, respec-
tively, with root-mean-square deviations for bond lengths and
angles of 0.007 Å and 1.041°, respectively. The Ramachandran plot
data indicated that 97.2% of the main-chain dihedral angles were
b
Rmerge =∑(I−bI>)/∑bI>, where I is the intensity measurement for a given re-
flection and bI> is the average intensity for multiple measurements of this reflection.
c
Rwork =∑|Fobs −Fcal|∑Fobs, where Fobs and Fcal are observed and calculated struc-
ture factor amplitudes.
d
R
free value was calculated for Rwork, using only an unrefined randomly chosen sub-
set of reflection data (5%).

K. Yamamoto et al. / Biochimica et Biophysica Acta 1820 (2012) 1469–1474
1471
found to be in the preferred regions, 2.8% in the allowed regions,
and 0% in the outlier regions.
156–167), α7 (residues 179–191), and α8 (residues 195–215). As in the
case of other GSTs, the structure of bmGSTD adopted the canonical GST
fold.
3
.2. Structural features of bmGSTD
3.3. Active-site residues
The amino acid sequence of bmGSTD shared 51.9% and 47.6% se-
quence identity with dipteran Delta-class GSTs, A. gambiae agGSTd1-6
25] and A. dirus adGSTD4-4 [26], respectively (Fig. 1A). The crystal struc-
In analogy with agGSTd1-6 [25], the GSH-binding site (G-site)
[
was located in the N-terminal domain of each monomer of the pre-
sent enzyme (Fig. 1B). In the G-site, 1 molecule of the inhibitor mol-
ecule S-hexyl glutathione (GTX) can be bound inside the pocket
formed by residues Ser11, Gln51, His52, Val54, Glu66, Ser67, and
Arg68 of bmGSTD (Fig. 2), which resemble the G-site residues
Ser9, His50, Ile52, Glu64, Ser65, and Arg66 in agGSTd1-6. The G-
site amino acid residues are mainly hydrophilic and have affinity
to the GSH moiety of the inhibitor. In bmGSTD, the 4 sites, i.e., the
side chain of Glu66, the hydroxyl group of Ser67, its main-chain
amide, and the side chain of Arg68, can form hydrogen bonds with
the γ-glutamyl region of GTX (Fig. 3A). Moreover, the main-chain
carbonyl and side chain of Val54, as well as the hydroxyl group of
Ser11, of the enzyme may interact with the cysteinyl moiety of
ture of bmGSTD was determined at 2.0-Å resolution and solved by the
molecular replacement method with agGSTd1-6 as the search model.
The obtained structure revealed a homodimer of the bmGSTD molecule
after analysis by PISA in REFMAC. Structural elements characterized by
the DSSP program showed that bmGSTD includes 8 α-helices and 4 β-
strands (Fig. 1A). Two distinct domains, N-terminal (residues 1–78) and
C-terminal (residues 89–216), as well as a linker part (residues 79–88)
in between these, were apparent (Fig. 1B). The N-terminal domain con-
tained 4 β-strands (β1 [residues 4–7], β2 [residues 12–23], β3 [residues
56–59], and β4 [residues 62–64]) and 3 α-helices (α1 [residues 12–23],
α2 [residues 43–48], and α3 [residues 67–78]). The C-terminal domain
consisted of α4 (residues 89–119), α5 (residues 126–142), α6 (residues
Fig. 1. Primary (A) and tertiary structure (B) of bmGSTD. (A) The sequence of bmGSTD was aligned with those of agGSTd1-6 and adGSTD4-4 (PDB IDs: 1PN9 and 3F63, respectively).
In this alignment, secondary structures were featured after being defined by the DSSP program; α-helices and β-strands are colored in orange and green, respectively. (B) The N-
and C-termini are labeled N and C, respectively. Starting positions of the α-helices and β-strands are marked α and β, respectively. The red and blue circles represent the G-site and
H-site, respectively. The figure was drawn with PyMOL (http://pymol.sourceforge.net).

1472
K. Yamamoto et al. / Biochimica et Biophysica Acta 1820 (2012) 1469–1474
Fig. 2. Amino acid residues responsible for the G-site of bmGSTD. GTX is shown in ma-
genta. Color scheme: oxygen, red; nitrogen, blue; and sulfur, yellow.
the inhibitor (Fig. 3B). Finally, the side chains of His52 and Gln51
can form hydrogen bonds with the glycyl portion of GTX (Fig. 3C).
Similar to bmGSTu [15], the current bmGSTD contained the
hydrophobic-binding site (H-site) in the C-terminal domain (Fig. 1B).
We compared the secondary structures at the H-site in bmGSTD with
those in agGSTd1-6 and adGSTD4-4 (cf. Fig. 1A), and the crystal structure
with that of agGSTd1-6. Results indicated that Tyr107, Tyr115, Phe119,
Phe206, and Ser212 in the H-site of bmGSTD established contact to
form a complex between GSH and the hydrophobic substrate.
To determine whether the above-indicated, seemingly important
residues exert real functions in bmGSTD enzyme activity, 5 residues
were each changed to Ala by site-directed mutagenesis, and the
resulting mutants were named S11A, Q51A, H52A, S67A, and R68A.
After purification from E. coli, each final preparation showed a single
band upon SDS‐PAGE. The kinetic parameters of bmGSTD mutants
were compared to those of the wild-type enzyme using CDNB and
m
GSH (Table 2). For both of the substrates, K values showed no
marked changes, while Vmax was decreased in particular in S11A
and S67A. The most prominent change was the decrease in kcat/K
m
values in all of the mutants so far tested.
3
.4. Motifs for stabilization of bmGSTD structure
An intersubunit motif (lock-and-key) was reported in the dimer
interface of adGSTD4-4, a Delta-class GST [27], wherein the “key” res-
idues (Glu65, Arg67, and Phe104) are inserted into a hydrophobic
pocket (Ala68, Leu103, Phe104, and Val107), the “lock” of the neigh-
boring subunit. Since bmGSTD has Glu66, Arg68, Ala69, Leu99,
Tyr100, and Ile103 in its amino acid sequence, it may possibly con-
serve the lock-and-key system, and a putative model for this is illus-
trated in Fig. 4.
Fig. 3. G-site structure of bmGSTD. The bmGSTD and GTX are shown in green and ma-
genta, respectively. Amino acid residues interact with the γ-glutamyl region of GTX
(A), cysteinyl moiety of GTX (B), and glycyl portion of GTX (C). Hydrogen bonds are in-
dicated by broken lines. Color scheme: oxygen, red; nitrogen, blue; and sulfur, yellow.
In adGSTD4-4, Leu6, Thr31, Leu33, Ala35, Glu37, Lys40, and Glu42
form a small hydrophobic core and an ionic bridge contributing to the
stabilization of the α2 helix [28]. The corresponding residues in
bmGSTD are Val8, Val33, Leu35, His37, Glu39, Lys42, and Gln44
4. Discussion
Here, we describe the structure of a lepidopteran GSTD for the first
time. In our previous study, bmGSTD had been misclassified into the
Theta class, for it appeared to have a role in the defense mechanisms
against oxidative stress and in the metabolism of lipid peroxidation
products [8,29]. The overall structure of bmGSTD, however, reveals
(
Fig. 1A). The latter 3 seemed to constitute the unique ionic bridge
motif comprising 3 charged residues in insect Delta-class GSTs, e.g.,
Glu37, Lys40, and Glu42 in adGSTD4-4 [28].

K. Yamamoto et al. / Biochimica et Biophysica Acta 1820 (2012) 1469–1474
1473
Table 2
Comparison of kinetic data.
conserved. Amino acid residues interacting with 4-hydroxynonenal
4HNE) have been proposed in Alpha-class GSTs from mice and humans
(
Enzyme
CDNB
GSH
[32,33]. These residues are not highly conserved in bmGSTD either, al-
though bmGSTD was able to conjugate GSH to 4HNE [8]. Understanding
the molecular basis of bmGSTD-mediated detoxification will require co-
crystallization of bmGSTD with a lipid peroxidant-GSH conjugate. Efforts
along these lines are currently being made and will provide more detailed
information regarding the antioxidant activity of this enzyme.
a
b
c
a
b
c
K
m
V
max
k
cat/K
m
K
m
V
max
kcat/K
m
Wild type
S11A
Q51A
H52A
S67A
R68A
0.48
0.21
0.60
1.22
1.39
NA
5.31
0.03
0.35
0.89
0.1
268
0.42
14.2
17.6
1.69
NA
0.52
1.19
2.33
0.68
2.7
4.25
0.05
0.55
0.43
0.09
NA
198
0.10
5.69
15.2
0.8
The bmGSTD protein possesses residues, resembling those previ-
ously proposed, to provide a subunit assembly system (lock-and-
key). The aromatic residue (Tyr100) was inserted into the hydro-
phobic pocket (Fig. 4). A small hydrophobic core and an ionic bridge
were found in the bmGSTD (cf. Results and Fig. 4). Arg68 was also
part of the lock-and-key motif; the mutation of this residue, which
leads to total loss of activity, might thus result in failure of dimer
formation. Indeed, when a single amino acid is replaced in this
motif of adGSTD4-4, catalytic efficiencies are altered [27]. Such an
intersubunit architecture appears to be highly conserved across
insect-specific and other GST classes [27]. Recently, 3 regions in-
volved in the folding, stabilization, and dimerization have been pro-
posed in adGSTD4-4 and its isoform, adGSTD3-3 [34]. We found that
these were not highly conserved in bmGSTD, suggesting that there
is variation in the corresponding amino acid residues among
Delta-class GSTs.
Similar to other-class GSTs, bmGSTD has GSH-conjugation activity
toward CDNB. To verify the participation of amino acid residues in
GSH binding, site-directed mutagenesis of 5 residues was performed
to map the active site of bmGSTD. With the resulting mutants, kinetic
analysis was carried out using CDNB and GSH as substrates. Results
suggest that all residues tested in bmGSTD could play important
roles in catalysis.
NA
NA
NA
Data are means of 3 independent experiments. NA, no activity.
a
Expressed in units of mM.
Expressed in units of μmol/mg/min.
Expressed in units of min
b
c
−1
−1
mM
.
similarities with other known insect Delta-class GSTs, which are di-
meric proteins with a globular shape. The X-ray data of bmGSTD
showed that each subunit consists of an N-terminal domain providing
the G-site and a C-terminal domain including the H-site. Identities in
the N-terminal and C-terminal domains between bmGSTD and agGSTd1-
6
were 64 and 48%, respectively, whereas those between bmGSTD and
adGSTD4-4 were 65 and 39%, respectively. This result is compatible
with the fact that the C-terminal domain is more variable, resulting in dif-
ferences in substrate specificity (this point will be debated below). The N-
terminal domain contains 3 α-helices and 4 β-strands, which together
form a βαβαββα motif. Similar to other GSTs, β3 and β4 are antiparallel
and connected by helix α1. The structure-based sequence alignment
(
Fig. 1A) showed that α6 in bmGSTD is smaller, whereas β8 in bmGSTD
is larger than those in mosquitoes. One α-helix is present between α3
and α4 in adGSTD4-4, which is absent in bmGSTD and agGSTd1-6.
As detailed above, the bound GSH could be located in the G-
site of the N-terminal domain of bmGSTD, which had similar sec-
ondary elements to those of agGSTd1-6, especially at the G-site
comprising Gln51, His52, Val54, Glu66, Ser67, and Arg68. These
residues form multiple hydrogen bonds with the glutamyl, cyste-
inyl, and glycyl residues of GSH. Previously, we proposed that
Ser11 in bmGSTD affected enzymatic activity [8]. In the current
structure model, the hydroxyl group of Ser11 makes close contact
with the sulfur atom of GSX (see Fig. 3B). Similar results have
been obtained in Glycine max; the cysteine residue of Tau-class
GSH points toward α-helix H2 and away from the catalytic Ser13
in this organism [30]. A serine residue at the N-terminal region
is also catalytically essential among Sigma-class GSTs, whereas ty-
rosine and cysteine are substituted by serine in the N-terminus of
Sigma-class and Omega-class GSTs of B. mori [9–11]. Overall, all G-
site residues are located in the N-terminal domain. The G-site of
bmGSTD consists of various hydrophobic residues, implying a com-
mon GSH-binding mechanism.
In summary, we described a high-resolution (2.0 Å) crystal struc-
ture of bmGSTD, representing the first structure of Delta-class GST
from lepidopteran insects. Its structure was comparable to that of
other GSTs. Site-directed mutagenesis targeting 5 residues (Ser11,
Gln51, His52, Ser67, and Arg68) showed notably reduced catalytic ef-
ficiency for CDNB and GSH, suggesting that, in bmGSTD, these resi-
dues play important roles in catalysis.
In contrast to well-conserved secondary structures, sequence
identities were relatively low among the 3 GSTs (Fig. 1A), particularly
in the C-terminal domain, which harbors the H-site. H-site sequence
variations are responsible for varying substrate specificity. The H-
site of agGSTd1-6 contains residues that are mainly hydrophobic:
Leu6, Ala10, Pro11, Leu33, Met34, Tyr105, Phe108, Tyr113, Ile116,
Phe117, Phe203, and Phe207 [26]. In bmGSTD, we found that 9 out
of 12 residues (Leu5, Ala12, Pro13, Leu35, Tyr107, Phe110, Tyr113,
Phe119, and Phe206) were identical. On the other hand, H-site resi-
dues of adGSTD4-4 (Tyr111, Tyr119, Phe123, Phe212, and Tyr215)
[31] were highly conserved. These residues correspond to Tyr107,
Tyr115, Phe119, Phe206, and Ser212 in bmGSTD, respectively. The
position of Tyr215 in adGSTD4-4 varies across insect species [31]. In
addition, the putative hydrophobic substrate-binding site of adGSTD4-4
Fig. 4. Representation of a model for subunit assembly via the lock-and-key motif in
bmGSTD. The key residues in one subunit of bmGSTD (Glu66, Arg68, and Tyr100) are
shown as green sticks. The other subunit is represented as a gray surface and the
lock residues are colored in yellow (Ala69), orange (Leu99), magenta (Tyr100), and
blue (Ile103).
(
Thr174, Phe212, Arg214, Tyr215, and Phe216) has been characterized
26]. Corresponding residues in bmGSTD may be Ser167, Phe206,
Asn211, Ser212, and Met213, suggesting that this site is not well
[

1
474
K. Yamamoto et al. / Biochimica et Biophysica Acta 1820 (2012) 1469–1474
15] Y. Kakuta, K. Usuda, T. Nakashima, M. Kimura, Y. Aso, K. Yamamoto, Crystallo-
[
Acknowledgements
graphic survey of active sites of an unclassified glutathione transferase from
Bombyx mori, Biochim. Biophys. Acta 1810 (2011) 1355–1360.
This work was supported in part by a Grant-in-Aid for Scientific Re-
search (KAKENHI; 21780049) from the Ministry of Education, Science,
Sports and Culture of Japan and by the Mishima Kaiun Memorial Foun-
dation. This work was also supported in part by the Research Grant for
Young Investigators from the Faculty of Agriculture, Kyushu University.
[16] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscilla-
tion mode, Methods Enzymol. 276 (1997) 307–326.
[
17] A. Vagin, A. Teplyakov, An approach to multi-copy search in molecular replace-
ment, Acta Crystallogr. D Biol. Crystallogr. 56 (2000) 1622–1624.
[18] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular struc-
tures by the maximum-likelihood method, Acta Crystallogr. D Biol. Crystallogr.
53 (1997) 240–255.
[
19] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta
Crystallogr. D Biol. Crystallogr. 60 (2004) 2126–2132.
References
[20] A.A. Vaguine, J. Richelle, S.J. Wodak, SFCHECK: a unified set of procedures for eval-
uating the quality of macromolecular structure-factor data and their agreement
with the atomic model, Acta Crystallogr. D Biol. Crystallogr. 55 (1999) 191–205.
[21] E. Krissinel, K. Henrick, Inference of macromolecular assemblies from crystalline
state, J. Mol. Biol. 372 (3) (2007) 774–797.
[1] I. Listowsky, M. Abramovitz, H. Homma, Y. Niitsu, Intracellular binding and trans-
port of hormones and xenobiotics by glutathione S-transferase, Drug Metab. Rev.
19 (1998) 305–318.
[
[
[
[
2] R.N. Armstrong, Structure, catalytic mechanism, and evolution of the glutathione
transferases, Chem. Res. Toxicol. 10 (1997) 2–18.
3] B. Mannervik, P.G. Board, J.D. Hayes, I. Listowsky, W.R. Pearson, Nomenclature for
mammalian soluble glutathione transferases, Methods Enzymol. 401 (2005) 1–8.
4] C.P. Tu, B. Akgül, Drosophila glutathione S-transferases, Methods Enzymol. 401
[22] W. Kabsch, C. Sander, Dictionary of protein secondary structure: pattern recognition of
hydrogen-bonded and geometrical features, Biopolymers 22 (1983) 2577–2637.
[23] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of
bacteriophage T4, Nature 227 (1970) 680–685.
[24] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferases, J. Biol. Chem. 249
(1974) 7130–7139.
(2005) 204–226.
5] X. Li, M.A. Schuler, M.R. Berenbaum, Molecular mechanisms of metabolic resis-
tance to synthetic and natural xenobiotics, Annu. Rev. Entomol. 52 (2007)
[25] L. Chen, P.R. Hall, X.E. Zhaou, H. Ranson, J. Hemingway, E.J. Meehan, Structure of
an insect δ-class glutathione S-transferase from a DDT-resistant strain of the
2
31–253.
6] H. Ranson, J. Hemingway, Mosquito glutathione transferases, Methods Enzymol.
01 (2005) 226–241.
malaria vector Anopheles gambiae, Acta Crystallogr.
(2003) 2211–2217.
[26] T. Lerksuthirat, A.J. Ketterman, Characterization of putative hydrophobic sub-
strate binding site residues of a Delta class glutathione transferase from Anopheles
dirus, Arch. Biochem. Biophys. 479 (2008) 97–103.
D Biol. Crystallogr. 59
[
[
4
7] R. Sawicki, S.P. Singh, A.K. Mondal, H. Benesc, P. Zimniak, Cloning, expression and
biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class
(
GST-1) glutathione S-transferases from Drosophila melanogaster, and identifica-
tion of additional nine members of the Epsilon class, Biochem. J. 370 (2003)
61–669.
[27] J. Wongsantichon, A.J. Ketterman, An intersubunit lock-and-key ‘Clasp’ motif in the
dimer interface of Delta class glutathione transferase, Biochem. J. 394 (2006) 135–144.
[28] A. Vararattanavech, P. Prommeenate, A.J. Ketterman, The structural roles of a con-
served small hydrophobic core in the active site and an ionic bridge in domain I of
Delta class glutathione S-transferase, Biochem. J. 393 (2006) 89–95.
[29] M. Hermes-Lima, T. Zenteno‐Savín, Animal response to drastic changes in oxygen
availability and physiological oxidative stress, Comp. Biochem. Physiol. 133C
(2002) 537–556.
6
[
8] K. Yamamoto, P. Zhang, F. Miake, N. Kashige, Y. Aso, Y. Banno, H. Fujii, Cloning, ex-
pression and characterization of theta-class glutathione S-transferase from the
silkworm, Bombyx mori, Comp. Biochem. Physiol. 141B (2005) 340–346.
9] K. Yamamoto, P. Zhang, Y. Banno, H. Fujii, Identification of a sigma-class glutathi-
one S-transferase from the silkworm, Bombyx mori, J. Appl. Entomol. 130 (2006)
[
5
15–522.
10] K. Yamamoto, H. Fujii, Y. Aso, Y. Banno, K. Koga, Expression and characteriza-
tion of sigma-class glutathione S-transferase of the fall webworm,
[30] I. Axarli, P. Dhavala, A.C. Parageorgiou, N.E. Labrou, Crystal structure of Glycine max glu-
tathione transferase in complex with glutathione: investigation of the mechanism op-
erating by the Tau class glutathione transferases, Biochem. J. 422 (2009) 247–256.
[31] J. Wongsantichon, R.C. Robinson, A.J. Ketterman, Structural contributions of Delta class
glutathione transferase active-site residues to catalysis, Biochem. J. 428 (2010) 25–32.
[32] L.M. Balogh, I.L. Trong, K.A. Kripps, L.M. Shireman, R.E. Stenkamp, W. Zhang, B.
Mannervik, W.M. Atkins, Substrate specificity combined with stereopromiscuity
in glutathione transferase A4-4-dependent metabolism of 4-hydroxynonenal,
Biochemistry 49 (2010) 1541–1548.
[
[
[
[
[
a
Hyphantria cunea, Biosci. Biotechnol. Biochem. 71 (2007) 553–560.
11] K. Yamamoto, S. Nagaoka, Y. Banno, Y. Aso, Biochemical properties of an
omega-class glutathione S-transferase of the silkmoth, Bombyx mori, Comp. Bio-
chem. Physiol. 149C (2009) 461–467.
12] K. Yamamoto, Y. Shigeoka, Y. Aso, Y. Banno, M. Kimura, T. Nakashima, Molecular
and biochemical characterization of a zeta-class glutathione S-transferase of the
silkmoth, Pesticide Biochem. Physiol. 95 (2009) 125–128.
[33] B. Xiao, S.P. Singh, B. Nanduri, Y.C. Awasthi, P. Zimniak, X. Ji, Crystal structure of a mu-
13] K. Yamamoto, H. Fujii, Y. Aso, Y. Banno, K. Koga, Expression and characterization
of a sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea,
Biosci. Biotechnol. Biochem. 71 (2) (2007) 553–560.
14] K. Yamamoto, H. Ichinose, Y. Aso, Y. Banno, M. Kimura, T. Nakashima, Molecular
characterization of an insecticide-induced novel glutathione transferase in silk-
worm, Biochim. Biophys. Acta 1810 (4) (2011) 420–426.
rine glutathione S-transferase in complex with a glutathione conjugate of
4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence
of signaling across the dimer interface, Biochemistry 38 (1999) 11887–11894.
[34] J. Piromjitpong, J. Wongsantichon, A.J. Ketterma, Differences in the subunit inter-
face residues of alternatively spliced glutathione transferases affects catalytic and
structural functions, Biochem. J. 401 (2007) 635–644.