Molecular characterization of an insecticide-induced novel glutathione transferase in silkworm

Article abstract of DOI:10.1016/j.bbagen.2011.01.003

Background: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated

Full text of DOI:10.1016/j.bbagen.2011.01.003

Biochimica et Biophysica Acta 1810 (2011) 420–426
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Molecular characterization of an insecticide-induced novel glutathione
transferase in silkworm
⁎
Kohji Yamamoto , Hirofumi Ichinose, Yoichi Aso, Yutaka Banno, Makoto Kimura, Takashi Nakashima
Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 August 2010
Received in revised form 23 December 2010
Accepted 7 January 2011
Available online 13 January 2011
Background: The glutathione transferase (GST) superfamily is involved in the detoxification of various
xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting
diazinon resistance and have investigated the enzyme properties of this GST.
Methods: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest
were changed to alanine by site-directed mutagenesis.
Results and conclusions: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST
belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly
conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze
the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic
analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function.
General significance: These results support the hypothesis that bmGSTu may play a role in insecticide
resistance in Bombyx mori.
Keywords:
Bombyx mori
Glutathione
Glutathione transferase
Lepidoptera
Site-directed mutagenesis
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
During the course of our studies to identify insecticide-induced
mRNAs in silkworms, we found that mRNA levels for sigma-class,
Glutathione conjugation is a step in a major pathway for the
detoxification of xenobiotics, as well as for the homeostasis of endogenous
compounds and results in the excretion of a water-soluble compound.
This reaction is catalyzed by glutathione transferases (GSTs) [EC 2.5.1.18],
which are widespread in both prokaryotes and eukaryotes [1,2]. There are
7 classes of GST (Alpha, Mu, Pi, Omega, Sigma, Theta and Zeta) in
mammals [3] and 6 classes of GST (Delta, Epsilon, Omega, Sigma, Theta
and Zeta) in dipteran insects, such as Anopheles gambiae and Drosophila
melanogaster [4]. Insect GSTs are investigated mainly from the viewpoint
of insecticide metabolism [5–7]. As Lepidoptera are the principal insect
pests in agriculture, knowledge of lepidopteran GSTs is of great
importance. We have previously characterized Sigma-, Omega-, Zeta-
and Delta-class GSTs in the domesticated silkworm, Bombyx mori, a
lepidopteran model insect [8–12], and a Sigma-class GST in the fall
webworm, Hyphantria cunea, one of the most serious lepidopteran pests
of broad-leaved trees [10].
omega-class and unclassified GSTs were induced. We previously
characterized sigma-class and omega-class GSTs [10,11]; however in
this article, we studied an unclassified B. mori GST which we have
called bmGSTu. In anticipation of a generally accepted designation for
the insect enzymes it should be pointed out that bmGSTu is a
temporary name. GSTs that fall into the unclassified group have
already been described for dipteran insects [13,14], although the
physiological functions of unclassified GSTs are uncertain. We
investigated certain enzyme properties using recombinant bmGSTu,
obtained by overexpression in Escherichia coli. The sequence of
bmGSTu and results of site-directed mutagenesis studies suggested
that the highly conserved Tyr, Ser and Asn residues in the N-terminal
region of bmGSTu are critical for enzyme catalysis. A large number of
agricultural insect pests are lepidopteran. As the silkworm is a model
animal for lepidopteran insects, it is useful to obtain information
concerning its detoxification capacity, in particular the novel GST
identified in the current study, for application to other pests.
2. Materials and methods
Abbreviations: CBB, Coomassie Brilliant Blue R250; CDNB, 1-chloro-2,4-dinitrobenzene;
ECA, ethacrynic acid; GSH, glutathione; GST, glutathione transferase; 4-HNE, 4-
hydroxynonenal; IPTG, isopropyl 1-thio-β-D-galactoside; 4-NPA, 4-nitrophenyl acetate;
LB, Luria–Bertani medium; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase
PCR; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate
2.1. Insects and tissue dissection
Larvae of the silkworm, B. mori, were reared on mulberry leaves in
the Institute of Genetic Resources, Kyushu University Graduate School
(Fukuoka, Japan). For toxicity tests, day-1 fifth-instar larvae were
exposed via direct contact of the larval abdomen with a diazinon
⁎
Corresponding author. 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 © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.01.003

K. Yamamoto et al. / Biochimica et Biophysica Acta 1810 (2011) 420–426
421
acetone solution (1 μg/g larva). Mortality was recorded 24-h post
treatment (day 2) and fat bodies from the larvae were dissected on ice
and stored at −80 °C until use. Total RNA was rapidly extracted from
the dissected fat bodies with an RNeasy Plus Mini Kit (Qiagen,
Valencia, USA) according to the manufacturer's instructions and was
then used for quantitative PCR or RT-PCR.
operations described below were conducted at 4 °C. The supernatant
was clarified by centrifugation at 10,000g for 15 min and subjected to
ammonium sulfate fractionation. The pellet obtained by 30% to 70%
salt saturation was resuspended in a 10 mM sodium phosphate buffer,
pH 8.2. After dialysis against the same buffer, the specimen was
subjected to anion-exchange chromatography on a DEAE-Sepharose
(GE Healthcare) column, and eluted with a linear gradient of 0 to
0.3 M NaCl. Fractions containing the enzyme were pooled, concen-
trated using a centrifugal filter (Millipore) and applied to a Superdex
200 column (GE Healthcare), and equilibrated with the same buffer
containing 0.2 M NaCl. An SDS-PAGE was performed using a 15%
polyacrylamide slab gel containing 0.1% SDS according to the method
of Laemmli (1970) [16]. Protein bands were visualized by staining
with Coomassie Brilliant Blue R250 (CBB). Protein concentration was
measured using a Protein Assay Kit (Bio-Rad Laboratories) with
bovine serum albumin as a standard.
2.2. Cloning and sequencing of the cDNA encoding bmGSTu
Total RNA was analyzed by RT-PCR. First-strand cDNA was produced
using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, USA)
and an oligo-dT primer. The resulting cDNA was used as a PCR template
with the following oligonucleotide primers: 5′-GGGCATATGTCTTCCT-
TAAAATTATACCAT-3′ (sense) and 5′-GGATCCTTATTGTTTGATATTCTT-
TTT-3′ (antisense). The primer designs were based on a partial sequence
obtained from the SilkBase EST database [15]. The underlined and
double-underlined regions are NdeI and BamHI restriction enzyme sites,
respectively, which were used to insert the PCR product into an
expression plasmid. PCR was performed for 1 cycle at 94 °C for 2 min,
then for 35 cycles at 94 °C for 1 min, 51 °C for 1 min and 72 °C for 2 min,
followed by 1 cycle at 72 °C for 10 min. The bmGSTu cDNA (bmgstu) was
ligated into the pGEM-T Easy Vector (Promega) and transformed into
E. coli JM109. The clone was sequenced in both directions using a
Thermo Sequenase Cycle Sequencing Kit (USB) and an automated DNA
sequencer (LIC-4200, Aloka). To obtain a complete sequence of bmgstu
and to deduce its amino acid sequence, DNASIS software (ver. 3.4) was
used. Homology alignment was performed using ClustalW (ver. 1.83),
with 10 and 0.2 as the gap creation penalty and the gap extension,
respectively. The phylogenetic tree was generated using neighbor-
joining plot software (http://www-igbmc.u-strasbg.fr/Bioinfo/ClastulX/
Top.html).
2.5. Measurements of enzyme activity
Under standard assay conditions, GST activity was spectrophoto-
metrically measured using 1-chloro-2,4-dinitrobenzene (CDNB) and
glutathione (GSH) [17]. Briefly, 0.01 ml of a test solution was added to
1 ml of a 50 mM sodium phosphate buffer (pH 6.5) containing 5 mM
CDNB and 5 mM GSH as substrates. Changes in absorbance at 340 nm/
min were monitored at 30 °C and converted into moles CDNB
conjugated/min/mg protein using the molar extinction coefficient of
the resultant 2,4-dinitrophenyl-glutathione: ꢀ340 =9600 M−1 cm−1
.
When other substrates were used in the assay for GST under same
conditions described above, changes in absorbance per minute were
converted into moles the substrate conjugated/min/mg protein using
the molar extinction coefficient: ꢀ224 =13,750 M−1 cm−1 for 4-
hydroxynonenal (4-HNE), ꢀ270 =5000 M−1 cm−1 for ethacrynic acid
(ECA), and ꢀ400 =8300 M−1 cm−1 for 4-nitrophenyl acetate (4-NPA)
[18,19]. Kinetic parameters (Km and Vmax) were obtained from a
double-reciprocal plot of data generated by varying the concentra-
tions of CDNB or GSH. The protein amount was determined using a
Protein Assay Kit (Bio-Rad) and bovine serum albumin was used as a
standard protein.
2.3. Quantitative PCR analysis
Total RNA was extracted from the fat body, silk gland, midgut, ovary
and testis of B. mori using an RNeasy Plus Mini kit (Qiagen) according to
the manufacturer's instructions. cDNAs were prepared from 2 μg total
RNA with SuperScript II reverse transcriptase (Invitrogen) and an oligo-
dT primer. Two sets of primers for bmGSTu and one set for ribosomal
protein 49 (rp49) were designed. The primer sequences were as
follows: bmGSTu forward primer, 5′-TGCGTACCACAAGGCTTTAGATT-3′
and bmGSTu reverse primer, 5′ -CCAGGTTCACTTCCGTCCA-3′ (111 bp
product); rp49 forward primer, 5′-CATTGGTTACGGTTCCAACAAG-3′
and rp49 reverse primer, 5′-GCATCATCAAGATTTCCAGCTC-3′ (101 bp
product). Real-time PCR was performed on a Thermal Cycler Dice Real
Time System TP-800 instrument (TAKARA) using SYBR Premix Ex Taq™
(Takara). PCR amplification began with a 10-s denaturation step at 95 °C
and then 40 cycles of denaturation at 95 °C for 5 s, annealing at 55 °C for
20 s and extension at 72 °C for 20 s. The samples were analyzed in
triplicate, and bmGSTu levels were normalized against corresponding
rp49 levels and expressed as the bmGSTu/rp49 ratio as described
previously [12]. These experiments were performed three times.
2.6. Site-directed mutagenesis
Amino acid-substituted mutants of bmGSTu were constructed
using the QuickChange Site-Directed Mutagenesis Kit according to the
manufacturer's recommendations (Agilent Technologies, Wilming-
ton, USA). The expression plasmid containing bmgstu was used as a
template. Full-length mutant cDNAs were systematically checked by
DNA sequencing.
3. Results
3.1. Sequence of bmgstu
Diazinon is widely used worldwide as an organophosphate
insecticide, and its use has promoted insect species that have acquired
high resistance to this substance [20]. Previously, we measured the LD50
values for diazinon in various strains of B. mori. The value in the most
resistant R1 strain was 12.5 μg/g larvae, which was about 6.6-fold
greater compared with the most susceptible S1 strain. Comparison
between these strains of mRNA levels encoding various proteins,
including GSTs, was then performed by microarray analysis using total
RNA from the fat bodies of day-2 fifth-instar larvae that had been
exposed to diazinon for 24 h. We found that, in the R1 strain, one GST
was induced compared with the control, which was not treated with
diazinon, whereas there was no significant induction in the S1 strain
after exposure to diazinon (Yamamoto et al., unpublished results). A
BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the Swiss-
2.4. Overexpression and purification of recombinant protein
The bmgstu clone, cloned in the pGEM-T Easy Vector, was digested
with NdeI and BamHI, subcloned into the expression vector, pET-11b
(Novagen), and transformed into competent E. coli Rosetta (DE3)
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 reach a final concentration of 1 mM to induce the production
of recombinant protein. After further incubation for 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

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K. Yamamoto et al. / Biochimica et Biophysica Acta 1810 (2011) 420–426
Prot database (http://expasy.org/sprot/) showed that the sequence of
the hybridized probe corresponded to the unclassified GSTs from Aedes
aegypti and A. gambiae.
When 4-HNE, ECA, or 4-NPA was used as alternative GSH-conjugating
substrates (instead of CDNB), bmGSTu activity was undetectable
(Table 1). The kinetic parameters of bmGSTu were evaluated using
CDNB and GSH as substrates (Table 2). The Km value for CDNB was
0.39 mM, which is comparable to Km values of Delta- and Omega-class B.
mori GSTs (0.48 and 0.67 mM, respectively), and about 25% of the value of
Sigma-class GSTs (1.57 mM) (previous values are cited from 8 to 11).
The inhibitory effects of model insecticides on bmGSTu were
examined with CDNB as a substrate. Results from inhibition experi-
ments of recombinant bmGSTu with diazinon, permethrin, deltame-
thrin and imidacloprid are shown in Fig. 6. Residual activities
decreased similarly with increasing amounts of each insecticide. In
the presence of 0.1 mM imidacloprid or deltamethrin, bmGSTu
showed 40% of its original activity. Diazinon caused the most
inhibition at a concentration of 0.1 mM, where bmGSTu activity was
decreased to 20% of its original activity. Although little information
about the inhibitory effects of these insecticides on GST is available,
previous reports indicate that GSTs participate in resistance to various
organophosphate compounds and pyrethroids in insect species such
as Haemaphysalis longicornis and Rhipicephalus appendiculatus [21–
23]. Activity of GSTs in these species was reduced to 85% and 60%,
respectively, by 0.1 mM deltamethrin [24], while bmGSTO exhibited
about 20% of its maximum activity under the same conditions [11].
In this study, we cloned the cDNA encoding this GST by RT-PCR
using total RNA from the fat bodies of the R1 strain. The nucleotide
sequence of bmgstu was deposited in GenBank under accession no.
AB561088. To confirm the induction described above, expression of
bmGSTu message was investigated by quantitative PCR using total
RNA from the fat bodies prepared as described above. The amount of
amplified product in the R1 strain was about 2.5-fold higher than that
in the control, whereas bmGSTu mRNA was induced by 0.8-fold after
treatment of diazinon in the S1 strain (Fig. 1).
The deduced amino acid sequence showed 34.4% and 33.5%
identity to GSTs from A. aegypti (AeGSTx2) and A. gambiae (AgGSTU3),
respectively, both of which belong to the unclassified group. These
amino acid sequences are aligned in Fig. 2. A phylogenetic tree,
generated from the amino acid sequences of the various insect GSTs
registered to date, indicate that the current protein is a novel member
of the B. mori unclassified GST group (Fig. 3). We named it bmGSTu
and the calculated molecular weight (24,457 Da) and isoelectric point
(7.80) of bmGSTu are similar to the values of other GSTs of B. mori
(Table 1).
3.2. Characterization of bmGSTu
3.3. Effect of Tyr7, Ser12 and Asn50 on bmGSTu activity
bmGSTu was overexpressed as a recombinant protein using an
E. coli expression vector. The product was soluble and was purified to
homogeneity by ammonium sulfate fractionation, anion-exchange
chromatography and gel-filtration. The final preparation produced a
single band following SDS-PAGE with a molecular weight of about
24,000 Da (lane 1, Fig. 4), which is close to the calculated value for the
deduced amino acid sequence (24,457 Da).
The enzyme properties of purified bmGSTu were studied with
CDNB and GSH as substrates. The pH optimum of bmGSTu was found
to be about 8 (Fig. 5A), which is similar to the optima of Delta- and
Zeta-class B. mori GSTs (Table 1). Fig. 5B shows that bmGSTu was
stable at temperatures below 50 °C, similar to Delta-, Omega-, and
Zeta-class B. mori GSTs (Table 1). As illustrated in Fig. 5C, bmGSTu
retained more than 75% of its original activity from pH 5 to 9, which is
a narrower pH range than those of other B. mori GSTs (Table 1).
Tyr, Ser, and sometimes Asn residues in the N-terminal half of GSTs
are highly conserved and catalytically essential. In bmGSTu, all 3
residues (Tyr7, Ser12, and Asn50) were present (Fig. 2). To see
whether any or all of these residues are critical for bmGSTu enzyme
activity, each residue was changed to Ala by site-directed mutagen-
esis. The resulting 3 mutants were named Y7A, S12A and N50A and
were purified from E. coli. Each final preparation showed a single band
upon SDS-PAGE (lanes 2, 3, and 4 of Fig. 4), and kinetic parameters for
CDNB and GSH were compared with those of the wild-type enzyme
(Table 2). For CDNB, the Km values of Y7A and S12A were increased
by 4- and 1.8-fold, respectively, while both Vmax values showed de-
creases of 87%. For GSH, the Km value of Y7A was augmented 2.6-fold,
but its Vmax was diminished to 8.5%. No large difference in Km value
was observed for S12A, although its Vmax fell to 8.0%. For both
S1 strain
1.0
R1 strain
1.0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
Fig. 1. Comparison of relative bmgstu mRNA levels between two strains of B. mori. Amounts of mRNA in fat bodies were analyzed by real-time PCR with probes detailed in Materials
and methods and plotted relative to rp49 mRNA levels. R1, the most resistant strain; S1, the most susceptible strain. Error bars denote SEM from three independent experiments.

K. Yamamoto et al. / Biochimica et Biophysica Acta 1810 (2011) 420–426
423
Fig. 2. Alignment of amino acid sequences of unclassified GSTs. Sequences of GSTs from different organisms were obtained from this study or the Swiss-Prot database (http://expasy.
org/sprot/): bmGSTu (the present study); AeGSTx1 (no. Q0C791); AeGSTx2 (no. Q95W09); AgGSTU3 (no. Q8MUQ3). Conserved amino acid residues are shaded.
substrates, the kcat/Km values of Y7A and S12A decreased to 14.8 to
20.5%, respectively. In summary, the most noteworthy findings were
the rises in the Km values of Y7A and S12A. For N50A, decreases were
seen in Vmax and kcat/Km values, although no marked changes in Km
were observed.
corresponded to silkworm GSTs and not to E. coli GST (Swiss-Prot
database: no. P0A9D2).
GSTs catalyze a broad range of reactions because different
members of the family exhibit varied substrate specificity. This also
seems to be true for B. mori GSTs, as seen in Table 1. Similar to the B.
mori Zeta-class GSTs, bmGSTu possessed no GSH-conjugation activity
toward 4-HNE. Therefore, bmGSTu might not participate in the
defense reaction to oxidative stress caused by lipid peroxidation,
which produces cytotoxic alkenals such as 4-HNE [31]. Like the B. mori
Omega- and Sigma-class GSTs, bmGSTu did not catalyze reactions
with 4-NPA and ECA. ECA, which can be catalyzed by B. mori Delta-
GST, is a known substrate for Alpha-, Mu- and Pi-class GSTs of
mammals. 4-NPA is a substrate for human Omega-class GST [32] but
not for bmGSTu or bmGSTO. Of note, natural substrates of bmGSTu are
so far unknown. However, the substrate specificity of bmGSTu seems
to be different from all known B. mori GSTs. Generally, a GST sequence
is divided into 2 regions called domain I and II for the N- and C-
terminal halves, respectively [2]. Domain I includes the GSH binding
site (G-site), and domain II has a hydrophobic substrate-binding site
(H-site). The diversity of H-site amino acids in the C-terminal region
of GSTs is responsible for substrate selectivity [2]. The C-terminal
regions of the B. mori GSTs are varied (sequence alignment not
shown), probably influencing their diverse substrate specificity.
The majority of highly conserved residues are present in the G-site.
Among these, Tyr (the 7th position in bmGSTu) is catalytically
essential for the Alpha-, Mu- and Pi-class GSTs, while Ser (the 12th
position in bmGSTu) is critical for the Delta-, Sigma-, Theta- and Zeta-
class GSTs [32–36]. In addition, another residue, Asn (the 50th
position in bmGSTu), was found to be important for Zeta-class GSTs
[29]. As described above, bmGSTu has all 3 residues, which were
targeted in the site-directed mutagenesis study. Among the resulting
mutants, Y7A gave the largest changes in Km as well as in Vmax and
kcat/Km values for CDNB and GSH. S12A showed some alterations in
Km, Vmax and kcat/Km. These results suggest that residues Tyr7 and
Ser12 in bmGSTu might play important roles in catalysis. N50A
resulted in changes in Vmax and kcat/Km, thus, N50A might contribute
to bmGSTu activity. In the crystal structure of the Anopheles dirus
Delta-class GST, 5 residues in the G-site (Ile52, Glu64, Ser65, Arg66
4. Discussion
To survey proteins associated with resistance to insecticides, we
screened for differential expression of GST mRNAs in insecticide-
resistant and susceptible strains of silkworms. bmgstu mRNA was
found to be preferentially expressed in the R1 strain which showed
the highest resistance to diazinon among the strains tested. We
focused on GST transcripts since various classes of GSTs contribute to
the detoxification of exogenous compounds. Similar results have been
obtained in other insect species: GST activities were higher in
organophosphate- and pyrethroid-resistant strains than in a suscep-
tible strain of fall armyworms [25] and elevated levels of GSTs were
associated with pyrethroid-resistance in mosquitoes [26–28]. More-
over, in soy, the Tau-class GST was induced in response to
diphenylether herbicide treatments [29]. The expressions of the
GSTs could lead to increasing rates of insecticide metabolism.
GSTs of the unclassified group were first described fairly recently
[13], probably due to the variety of GST genes discovered during
advanced genomic research. Of interest, no orthologs of unclassified
GSTs have been detected in D. melanogaster and Apis mellifera [30]. To
date, investigations of the molecular and biochemical properties of
unclassified insect GSTs are limited. A GST from A. aegypti (AeGSTx2)
has been overexpressed in E. coli and was purified from the soluble
fraction by His-tag chromatography, although other overexpressed
GSTs (AeGSTi1 and AeGSTx1) were retained in the insoluble fraction
[14]. In our E. coli production system for bmGSTu, we did not add any
tags to avoid potential side effects of the tag on the N-terminal region
of the protein and we could successfully purify bmGSTu and
characterize its enzymatic properties. To confirm that the proteins
were different from the endogenous E. coli GST, the N-terminal
sequences of purified GST mutants were determined. The sequences

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K. Yamamoto et al. / Biochimica et Biophysica Acta 1810 (2011) 420–426
(kDa)
Ag-sigma-P46428
P46428Anopheles.seq
M
1
M
2
M
3
M
4
1000
1000
250
150
P46437Musca.seq
Md-sigma-P46437
100
1000
75
P41043Dros.seq
Dm-sigma-P41043
50
37
Msex_P46429_.seq
Ms-sigma-P46429
Hc-sigma-Q4ACU7
1000
Bm-sigma-Q5CCJ4
Q5CCJ4silkmoth.seq
25
20
gaAmgb-ioameQeg8a6-DQ8856.sDe8q5
348
446
CG6662aa.seq
Dm-omega-CG6662
1000
755
CG6781_aa_.seq
Dm-omega-CG6781
CG6776aa.seq
Dm-omega-CG6776
990
CG6673PB_aa_.seq
Dm-omega-CG6773PB
1000
Dm-omega-CG6773PA
CG6673PA_aa_.seq
Fig. 4. Recombinant bmGSTu and site-directed bmGSTu mutants overexpressed in
E. coli. Purified recombinant proteins were subjected to SDS-PAGE followed by CBB
staining. M, protein size markers; lane 1, wild type; lane 2, Y7A; lane 3, S12A; lane 4,
N50A.
thBism-omega-A8R5V3
1000
999
silkzeta.seq
BmGSTZ
1000
Dr-zeta-Q6DGL3
Q6DGL3zebra.seq
Q9VHD3Dros.seq
Dm-zeta-Q9VHD3
In summary, we studied the biochemical properties of an unclassi-
fied GST (bmGSTu) that was overexpressed using mRNA from the R1
strain. Furthermore, we characterized which amino acid residues of
bmGSTu are related to catalytic efficiency by employing site-directed
mutagenesis and showed that 3 conserved residues, Tyr7, Ser12 and
Asn50 contributed to the catalysis of CDNB. On the basis of these
observations, we predict that bmGSTu might be one of the enzymes
involved in the xenobiotic detoxification system. It will be important to
compare these enzymes for expression rates, activities, substrate
specificity and resistance spectrum in different strains of B. mori. In
our previous differential expression study, we identified several
candidates, including cytochrome P450, esterase, and other GSTs,
which might be associated with insecticide resistance. The character-
ization of these properties will facilitate the development of molecular
markers and influence the practice of insecticide-resistance manage-
ment strategies for agricultural pests.
873
Q8MUQ5mosquito.seq
Ag-zeta-Q8MUQ5
1000
Q16P53_AeGSTi1.seq
AeGSTUi1-Q16P53
Q8MUQ6_AgGSTU1.seq
AgGSTU1-Q8MUQ6
Q8AMgGUSQT4U_A2-gQG8SMTUQ2.4seq
1000
1000
AeGSTx1-Q0C791
Q0C791_AeGSTx1.seq
ORF_aa_.seq
bmGSTU
676
1000
850
Q95W09_aeGSTx2.seq
AeGSTx2-Q95W09
Q8AMgGUSQT3U_A3-gQG8SMTUQ3.3seq
Q6B0mG-Kd5elstialk-Q.se6q0GK5
Ag-delta-Q93113
726
1000
P28338Musca.seq
Md-delta-P28338
999
1000
DM_P20432_.seq
Dm-delta-P20432
Fig. 3. Phylogenetic analysis of GST amino acid sequences. The phylogenetic tree was
made with neighbor-joining plot software using GST sequences obtained from NCBI
(http://www.ncbi.nlm.nih.gov/), Swiss-Prot (http://expasy.org/sprot/) and flybase
(http://flybase.org/). Each entry contains a species name, GST class, and accession
number. Numbers indicate branch length. Ag, A. gambiae; Md, Musca domestica, Dm,
D. melanogaster; Ms, Manduca sexta; Hc, Hyphantria cunea; Bm, B. mori; Ae, A. aegypti.
Numbers attached to nodes represent the bootstrap value.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research (KAKENHI; 21780049) from the Ministry of Education,
Science, Sports and Culture of Japan and by the Mishima Kaiun
Memorial Foundation. This work was also supported in part by the
National Bioresource Project (silkworm) from the above Ministry. The
authors are greatly indebted to Dr. Yuki Nakamura, Dr. Hiroaki Noda
and Dr. Kazuei Mita at the National Institute of Agrobiological Sciences
for a series of microarray analyses.
and Met101) were shown to interact with GSH [37]. Such crystallog-
raphy studies applied to bmGSTu, in addition to more detailed site-
directed mutagenesis experiments, will help identify the residues that
are crucial for catalytic reactions.
Table 1
Comparison of properties of GSTs determined in the present and in previous works.
Properties
bmGSTu
bmGSTD
bmGSTO
bmGSTS
bmGSTZ
Calculated molecular weight
Calculated isoelectric point
Optimum pH
24,457
7.80
4–9
24,225
8.35
8
29,806
6.01
7–8
23,338
5.79
8
24,727
8.5
6
Stable pH range
Stable temperature range
Substrate specificity
5–9
b50 °C
CDNB
3–9
2–12
4–11
b40 °C
CDNB
4–10
b50 °C
Dichloro acetic acid
b50 °C
CDNB
4-HNE
4-NPA
ECA
b50 °C
CDNB
4-HNE
Abbreviations used: bmGSTD, B. mori Delta-class GST; bmGSTO, B. mori Omega-class GST; bmGSTS, B. mori Sigma-class GST; bmGSTZ, B. mori Zeta-class GST. Data for these are cited
from Yamamoto et al. (2005, 2006, 2007, 2009a, b) [8–12].

K. Yamamoto et al. / Biochimica et Biophysica Acta 1810 (2011) 420–426
425
Table 2
Comparison of kinetic data.
A
100
75
50
25
0
Enzyme
CDNB
GSH
kcat/Km Km
Km
Vmax
Vmax
kcat/Km
23.7
3.79
4.88
Wild type 0.39 (0.06) 17.7 (0.01) 138
Y7A
S12A
N50A
3.94 (0.61) 30.5 (0.1)
10.4 (0.54) 2.60 (0.46)
3.14 (0.42) 2.45 (0.21)
1.58 (0.08) 2.18 (0.58)
0.70 (0.05) 2.29 (0.2)
0.24 (0.03) 2.51 (0.01) 115
21.0
20.4
3.70 (0.05) 3.83 (0.07) 11.5
Kinetic data were determined for CDNB and GSH. Values except those of kcat/Km are
expressed as mean (SD) of 3 independent experiments. Km and Vmax are expressed as
mM and μmol·mg−1·min−1, respectively. kcat/Km are expressed as min−1 mM−1
.
100
80
60
40
20
0
2
4
6
8
10
12
pH
B
100
75
50
25
0
-5
-4
-3
-2
-1
0
Log [Inhibitor] mM
Fig. 6. Effects of insecticides on the activity of bmGSTu. Enzymatic activity was
measured in the presence of various concentrations of insecticide: diazinon (closed
circle), permethrin (open square), deltamethrin (open circle) or imidacloprid (closed
square). The value from the assay with 1×10−5 M of insecticide was set to 100%. Data
represent averages with SD from 3 independent experiments.
20
40
60
80
100
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