S-Glutathionyl-(chloro)hydroquinone reductases: A novel class of glutathione transferases

Article abstract of DOI:10.1042/BJ20091863

Sphingobium chlorophenolicum completely mineralizes PCP (pentachlorophenol). Two GSTs (glutathione transferases), PcpC and PcpF, are involved in the degradation. PcpC uses GSH to reduce TeCH (tetrachloro-p- hydroquinone) to TriCH (trichlorop-hydroquinone)

Full text of DOI:10.1042/BJ20091863

Biochem. J. (2010) 428, 419–427 (Printed in Great Britain) doi:10.1042/BJ20091863
419
S-Glutathionyl-(chloro)hydroquinone reductases: a novel class of
glutathione transferases
Luying XUN*1, Sara M. BELCHIK*, Randy XUN*, Yan HUANG*, Huina ZHOU†, Emiliano SANCHEZ*, ChulHee KANG*
and Philip G. BOARD†
*School of Molecular Biosciences, Washington State University, Pullman, WA 99164–7520, U.S.A., and †John Curtin School of Medical Research, Australian National University,
Canberra, Australian Capital Territory, Australia
Sphingobium chlorophenolicum completely mineralizes PCP
(pentachlorophenol). Two GSTs (glutathione transferases), PcpC
and PcpF, are involved in the degradation. PcpC uses GSH to
reduce TeCH (tetrachloro-p-hydroquinone) to TriCH (trichloro-
p-hydroquinone) and then to DiCH (dichloro-p-hydroquinone)
during PCP degradation. However, oxidatively damaged PcpC
produces GS-TriCH (S-glutathionyl-TriCH) and GS-DiCH (S-
glutathionyl-TriCH) conjugates. PcpF converts the conjugates
into TriCH and DiCH, re-entering the degradation pathway. PcpF
was further characterized in the present study. It catalysed GSH-
dependent reduction of GS-TriCH via a Ping Pong mechanism.
First, PcpF reacted with GS-TriCH to release TriCH and formed
disulfide bond between its Cys53 residue and the GS moiety.
Then, a GSH came in to regenerate PcpF and release GS–
SG. A TBLASTN search revealed that PcpF homologues were
widely distributed in bacteria, halobacteria (archaea), fungi and
plants, and they belonged to ECM4 (extracellular mutant 4)
group COG0435 in the conserved domain database. Phylogenetic
analysis grouped PcpF and homologues into a distinct group, sep-
arated from Omega class GSTs. The two groups shared conserved
amino acid residues, for GSH binding, but had different residues
for the binding of the second substrate. Several recombinant PcpF
homologues and two human Omega class GSTs were produced in
Escherichia coli and purified. They had zero or low activities for
transferring GSH to standard substrates, but all had reasonable
activities for GSH-dependent reduction of disulfide bond (thiol
transfer), dehydroascorbate and dimethylarsinate. All the tested
PcpF homologues reduced GS-TriCH, but the two Omega class
GSTs did not. Thus PcpF homologues were tentatively named
S-glutathionyl-(chloro)hydroquinone reductases for catalysing
the GSH-dependent reduction of GS-TriCH.
Key words: glutathione conjugate, glutathione transferase
(GST), glutathione-dependent reductase, pentachlorophenol,
S-glutathionyl-hydroquinone.
back into TriCH and DiCH respectively [11]. When the pcpF
gene is disrupted, S. chlorophenolicum degrades PCP more
slowly and becomes more sensitive to PCP. Consequently, the
accumulation of the conjugates is detrimental to the bacterium.
Thus PcpF plays two roles: channelling the conjugates back to
the metabolic pathway and preventing the accumulation of the
harmful conjugates.
PcpC and PcpF fall into different classes of GSTs. The two
proteins share 17.7% sequence identity with a global alignment
score of −48. A BLASTP search shows the N-terminus of PcpC is
most similar to the Zeta class of GSTs and the C-terminus is highly
homologous with the Beta class of GSTs. PcpC has been assigned
to the Zeta class, but it does not have high sequence identity with
any GSTs. The closest relative is a hypothetical GST (GenBank®
accession number AAZ25344) of Colwellia psychrerythraea 34H
(27.3% sequence identity) as determined by a TBLASTPN search
of bacterial genomes. In contrast, PcpF is highly conserved in
bacteria and in Saccharomyces cerevisiae [12]. S. cerevisiae has
three PcpF homologues, and ECM4 (extracellular mutant 4), a
protein involved in cell-surface biosynthesis and architecture, is
the most similar to PcpF. Although ECM4 has been characterized
as an Omega class GST (GTO2) for its ability to use some
substrates of the Omega class GSTs [12], it shares less than 20%
INTRODUCTION
PCP (pentachlorophenol) was first synthesized in 1872 [1], but
its massive release into the environment started in the late
1930s, associated with its use as a wood preservative [2]. Since
then there has been no evidence of natural production of PCP,
although some bacteria have evolved the ability to degrade
it within the past 100 years. Sphingobium chlorophenolicum
ATCC39723 was isolated in 1985 [3]. The genes and enzymes
involved in the degradation pathway of PCP have been identified
and characterized from the bacterium (Figure 1) [4]. PcpB, a
flavin-containing mono-oxygenase, oxidizes PCP to tetrachloro-
p-benzoquinone, which can be chemically or enzymatically
reduced by PcpD to TeCH (tetrachloro-p-hydroquinone) [5,6].
PcpC, a GST (glutathione transferase), uses GSH to reductively
dechlorinate TeCH to TriCH (trichloro-p-hydroquinone) and
then to DiCH (2,6-dichloro-p-hydroquinone) [7,8]. PcpA, a
dioxygenase, cleaves the aromatic ring of DiCH to produce 2-
chloromalaylacetate, a relatively common metabolic intermediate
in the degradation of chlorinated aromatic compounds [9]. A
cysteine residue at the active site of PcpC can be oxidatively
damaged, and the damaged PcpC produces GS (S-glutathionyl)-
TriCH and GS-DiCH conjugates [10]. PcpC cannot transform
the conjugates, but PcpF converts the GS-TriCH and GS-DiCH
Abbreviations used: At-, Arabidopsis thaliana; CDNB, 1-chloro-2,4-dinitrobenzene; DHA, dehydroascorbate; DHAR, DHA reductase; DiCH, dichloro-
p-hydroquinone; DMAV, dimethylarsinate; DTT, dithiothreitol; Ec-, Escherichia coli; ECM4, extracellular mutant 4; GS-DiCH, S-glutathionyl-DiCH; GST,
glutathione transferase; GSTO, glutathione transferase Omega class; GS-TriCH, S-glutathionyl-TriCH; h, human; HED, 2-hydroxyethyl disulfide; LB,
Luria–Bertani; Ni-NTA, Ni2+-nitrilotriacetate; PCP, pentachlorophenol; Re-, Ralstonia eutropha; TeCH, tetrachloro-p-hydroquinone; TriCH, trichloro-p-
hydroquinone.
1
To whom correspondence should be addressed (email xun@mail.wsu.edu).
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L. Xun and others
Figure 1 Pentachlorophenol degradation pathway of S. chlorophenolicum ATCC 39723
PcpB, PCP 4-monooxygenase; PcpD, tetrachloro-p-benzoquinone reductase; PcpC, TeCH reductive dehalogenase; PcpC-ox, oxidatively damaged PcpC; PcpA, DiCH 1,2-dioxygenase; PcpE,
chloromaleylacetate reductase; PcpF, GS-(chloro)hydroquinone reductase.
sequence identity with other Omega class GSTs. The conserved
domain database has an ECM4 as group COG0435, which is not
assigned to the Omega class of GSTs [13] (COGs are assigned
for clusters of orthologous groups of proteins, and orthologues
occupy the same functional niche in different species).
In the present study, we determined that PcpF, ECM4 and
two bacterial PcpF homologues used GS-TriCH as a substrate,
whereas the two human Omega class GSTs did not. The
substrate difference and sequence analysis suggest that PcpF and
homologues, including ECM4, belong to a new class of GSTs:
S-glutathionyl-(chloro)hydroquinone reductases. The reaction
mechanism for GS-TriCH reduction was also investigated.
were confirmed by colony PCR and sequencing. The correct
clones were directly used for protein production. N-terminally
His6-tagged hGSTO (human glutathione transferase Omega class)
1 and hGSTO2-C4 {a stabilized variant that contains five cysteine
residue substitutions (C80S, C121S, C136S, C140S, C170S and
C214S) and a deletion of last four amino acid residues at the C-
terminus (Phe240, Gly241, Leu242 and Cys243); the modified enzyme
retains 70% of the dehydroascorbate reductase activity of the
wild-type enzyme [14]} were cloned in to the pQE30 vector with
E. coli M15 (carrying pRep4) as host as described previously.
Protein purification
E. coli stains carrying the cloned gene on an expression vector
were grown in 1 litre of LB medium at 37◦C to D = 0.6 at 600 nm,
induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside, and
then incubated at room temperature (23◦C) for 5 h. PcpF-C14S
was purified as described previously [11]. All other proteins
were purified with Ni-NTA (Ni2+-nitrilotriacetate)–agarose resin,
according to the manufacture’s instructions (Qiagen). Targeted
proteins were concentrated and the buffer was changed to
potassium phosphate buffer [40 mM potassium phosphate, pH 7,
containing 2 mM DTT (dithiothreitol) with Centriprep Ultracel
YM-10 (Millipore)]. Glycerol was added to 10% (v/v) before
storage at −80◦C.
EXPERIMENTAL
Chemicals and enzymes
All chemicals were obtained from Sigma–Aldrich or Fisher
Scientific. S. cerevisiae glutathione reductase was obtained from
Sigma–Aldrich. Restriction enzymes were obtained from New
England Biolabs. PCR was performed with Taq DNA polymerase
and primers from Invitrogen.
Bacterial strains, plasmids and culture conditions
Escherichia coli strains BL21(DE3) and M15, containing the
pRep4 (Qiagen) antibiotic resistance plasmid, were grown in
LB (Luria–Bertani) medium or on LB agar plates at 37◦C or as
specified. Kanamycin (30 μg/ml) was added to the LB medium
when required. The pET30-LIC plasmid carrying the pcpF-C53A
mutant gene has been described previously [11]. The genes coding
for PcpF, YqjG (GenBank® accession number NP_417573)
of E. coli, YqjG (GenBank® accession number YP_295669) of
Ralstonia eutropha (also known as Cupriavidus necator), JMP134
and ECM4 (GenBank® accession number NP_013002) of S.
cerevisiae were cloned into pET30-LIC with a C-terminal His6-
tag. The cloning was essentially the same as described previously
[11]. Briefly, genomic DNA was isolated with PureGene DNA
Isolation Kit (Gentra). The target gene was amplified from the
genomic DNA with designed primers (available upon request
from L.X.). The PCR product was digested by NdeI and HindIII
(or another restriction enzyme). The digested PCR products
were ligated into pET30-LIC vector (Novagen). The ligation
products were electroporated into E. coli BL21(DE3). Clones
Enzyme assays
GS-TriCH reduction was analysed by HPLC as previously
described [11]. For kinetic analysis, a spectrometric assay was
developed, using 1 ml of 70 mM potassium phosphate buffer,
pH 6.5, containing 50 μM GS-TriCH, 2 mM ascorbic acid,
5 mM GSH, 300 μM NADPH and 1 μl of glutathione reductase
(G3664–500UN; Sigma). The reaction was monitored at 340 nm.
Then, 1–5 μl of PcpF, or other GSTs, was added and the decrease
at 340 nm for NADPH consumption was continuously monitored.
The change in slope after PcpF addition was used to calculate the
enzymatic rate. For kinetic analysis, GSH was varied from 0.2 to
10 mM and GS-TriCH from 5 to 200 μM. GS-TriCH was prepared
by using ultracentrifuged E. coli cell extracts containing over-
produced PcpC-C14S in essentially the same manner as described
previously [11] with some modifications. Specifically, reactions
were carried out in 1 ml of 70 mM potassium phosphate buffer,
pH 6.5, containing 2 mM ascorbic acid, 0.5 mM GSH, 200 μM
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TeCH and 20 μl of PcpC-C14S (ultracentrifuged cell extracts of
overexpressing E. coli, 27.6 mg/ml). The sample was incubated
at 30◦C for 10 min and then heated at 65◦C for 10 min. The
quantitative conversion of TeCH into GS-TriCH was confirmed
by HPLC analysis as described previously [11]. The denatured
proteins were removed by centrifugation (20000 g for 2 min),
and the supernatant was stored at 4◦C, which was stable for at
least 1 week.
Similar spectrometry assays were used to analyse the kinetic
parameters with cysteine, 2-mercaptoethanol and DTT as co-
substrate rather than GSH. Again, the reactions were started and
NADPH consumption was monitored for 1 min. Then, PcpF was
added. The slope difference was used to calculate PcpF activity.
The GST activity was measured with 0.2 mM CDNB (1-
chloro-2,4-dinitrobenzene), or ethacrynic acid, as the substrate in
100 mM potassium phosphate buffer, pH 6.5, containing 0.2%
Triton X-100, and the continuous increase in absorbance at
340 nm (or 270 nm) was monitored [15]. Thiol transferase
activity was measured with HED (2-hydroxyethyl disulfide)
genome (nine in total) in the order of Halobacteriales. However,
no significant similarities were found in any other orders within
the archaea. In bacteria, PcpF had many homologues in the phyla
of actinobacteria, cyanobacteria, firmicutes (containing orders
of Bacillales, Clostridia and Lactobacillales) and proteobacteria.
The top 100 hits against bacterial genomes showed sequence
identity from 55% to 65%. In eukaryotes, PcpF homologues
were well conserved in fungi and plants, but not in animals. The
top 100 hits against fungal genomes showed sequence identity
from 36% to 50%. Some species had more than one homologue,
e.g. S. cerevisiae had three homologues, GTO1, ECM4 and GTO3
[12]. PcpF homologues were present in every sequenced plant
genome, and some plants had several PcpF homologues. For
example, Arabidopsis thaliana had four homologues (GenBank®
accession numbers, NP_199315, NP_001031671, NP_199312
and NP_568632). These conserved homologues had at least
30% sequence identity with PcpF by global alignment. PcpF
homologues were not found by TBLASTN search of the
sequenced genomes in most archaea, some bacteria and all animal
species.
as
a substrate [16]. DHA (dehydroascorbate) reductase
assay and DMAV (dimethylarsinate) reductase assay were
preformed using established protocols [17]. The S-(2ꢁ,4ꢁ-
dichlorophenacyl)glutathione reductase activity was assayed by
following the decrease in absorption at 276 nm as described
previously [18], except that the GSH concentration was increased
to 5 mM for all the assays except for with ethacrynic acid, which
had a very high non-enzymatic reactivity at 5 mM GSH. The rates
before and after enzyme addition were recorded. The difference
was used to calculate the enzyme activities.
Sequence comparison of PcpF with selected proteins by global
alignment
The proteins identified by TBLASTN were further compared
by global alignment. The R. eutropha JMP134 homologue [Re-
YqjG (R. eutropha YqjG) hypothetic protein] was the fourth most
homologous with PcpF (64% identity by TBLASTN) and the
E. coli homologue [Ec-YqjG (E. coli YqjG) hypothetic protein]
was not on the top-100 list of TBLASTN search of bacterial
genomes. Ec-YqjG was selected for further analysis because
it was from E. coli, a model organism. PcpF and Re-YqjG
shared 60.7% sequence identity with a global alignment score of
1355. The two YqjGs had 63% identity and a global alignment
score of 1455. PcpF and Ec-YqjG had 51.7% sequence identity
with a global alignment score of 1107. A. thaliana had four
PcpF homologues listed as ECM4-like proteins with sequence
identity ranging from 31.2 to 41.8% by global alignment. ECM4a
(GenBank® accession number NP_199315) of A. thaliana was
the most similar to PcpF, sharing 41.8% sequence identity with
a global alignment score of 845. S. cerevisiae had three PcpF
homologues, and ECM4 was the most homologous to PcpF with
35.4% sequence identity and a global alignment score of 625. As
the three yeast homologues are characterized as Omega class
GSTs [12], we compared the sequence of PcpF with that of
hGSTO1-1, the most characterized Omega class GST [21]. The
two proteins had a sequence identity of 20.1% with a negative
global alignment score of −15. PcpF and hGSTO2-2 shared only
18.6% sequence identity with a global alignment score of −24.
Furthermore, ECM4 and hGSTO1-1 had 15.7% sequence identity
with a global alignment score of −128.
Static light-scattering
The average molecular mass of individual GSTs was measured
by combined size-exclusion chromatography and multi-angle
laser light scattering. Briefly, 100 μg of GST in a freshly
prepared buffer (1× PBS) was loaded on to a BioSep-SEC-S2000
column (Phenomenex) and eluted isocratically with a flow rate
of 0.5 ml · min−1. The elute was passed, in tandem, through a UV
detector and a Dawn EOS laser light-scattering detector (Wyatt
Technology). Scattering results were analysed using the Zimm-
fitting method with software (ASTRA) provided by the instrument
manufacturer.
Protein sequence analysis
TBLASTN (searching a translated nucleotide database using
a protein query) was used to identify PcpF homologues in
sequenced genomes at the website of PubMed. Protein sequences
were aligned by using ClustalW [19] and a phylogenetic tree
was generated by using the neighbour-joining method of MEGA
version 4.0.2 [20]. The evolutionary distances were computed
using Poisson correction and were in units of the number of
amino acid substitutions per site. Optimal global alignment of two
protein sequences was performed with the ALIGN program
of Biology Workbench (http://workbench.sdsc.edu). All of the
analyses were performed with default parameters.
Phylogenetic analysis of GSTs with a catalytic cysteine residue
Sequence analysis is the primary basis for the classification of
GSTs. Well characterized classes include: Alpha, Mu, Pi and
Kappa of mammalian GSTs [22]; Phi, Tau, Lambda, and DHAR
(dehydroascorbate reductase) of plant GSTs [23]; Delta from
insects [24]; and Beta of bacterial origin. In addition, Theta, Zeta
and Omega classes of GSTs are widely present in a variety of
life forms [21,24]. The GSTs can also be grouped on the basis
of active-site residues [22]. The Alpha, Mu and Pi classes of
GSTs have an N-terminal tyrosine residue that interacts with
GSH to stabilize the thiolate anion; a serine residue fulfils the
function in the Theta, Zeta, Kappa, Phi and Tau classes [25];
RESULTS
BLAST search of PcpF homologues
BLAST searches revealed that PcpF had homologues in selected
species in all domains of life. A TBLASTN search of archaeal
genomes revealed the presence of PcpF homologues in every
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Multiple sequence alignment and amino acid residues in substrate
binding
Among the GSTs in the Omega, Lambda, DHAR and PcpF
homologues, only the structure of hGSTO1-1 is available. Three
PcpF homologues and hGSTO1-1 were aligned by ClustalW.
The secondary structure of hGSTO1-1 is presented for reference
(Figure 3). Despite the fact that hGSTO1-1 was much smaller
than PcpF homologues, the amino acid residues (Cys32, Phe34,
Lys59, Leu71, Val72, Pro73, Glu85 and Ser86; Figure 3, underlined
residues) involved in the binding of GSH in hGSTO1-1 were well
conserved in the other proteins. The amino acid residues (Ser121
,
Pro124, Ser125, Gly128, Ser129, Trp180, Arg183 and Trp222; Figure 3,
bold residues), positioned for the binding of the second substrate,
were not conserved between hGSTO1-1 and PcpF homologues,
except for Arg183, but the corresponding amino acid residues were
highly conserved among the three PcpF homologues (Figure 3,
bold residues). The variation in key residues for substrate binding
implies that PcpF homologues may have a different substrate
range from that of hGSTO1-1.
Figure 2 Dendrogram of inferred phylogenetic relationships among GSTs
with an N-terminal cysteine residue known or assumed to interact with the
thiol group of bound GSH
Recombinant enzyme production and purification
The pcpF gene was re-cloned as a C-terminally His6-tagged
protein that was over-produced as a major soluble protein in E.
coli. The over-produced protein was purified by Ni-NTA–agarose
resin. The His6 tag did not affect the specific activity of PcpF for
GS-TriCH reduction [11]. Thus the His6-tagged PcpF was used to
further characterize its activities. ECM4 has been characterized
as an Omega class GST [12], but phylogenetic analysis grouped
ECM4 with PcpF homologues rather than the Omega class
GSTs (Figure 2). In order to compare PcpF homologues with
the Omega class of GSTs, Re-YqjG, Ec-YqjG and ECM4 were
cloned in expression vector pET30-LIC, as C-terminally His6-
tagged proteins. The GSTs and hGSTO1-1 and hGSTO2-2 were
produced in E. coli as His6-tagged proteins and purified by Ni-
NTA–agarose resin.
The tree was constructed using the neighbour-joining method. The numbers on the
branches are bootstrap values (percentage of 1000 runs), indicating the frequency of
grouping in the cluster. The distance correlates to the number of amino acids substituted
®
per site. The GSTs and corresponding GenBank protein accession numbers are as
follows: Ec-YqjG, NP_417573; Re-YqjG, YP_295669; ECM4, NP_013002; PcpF, AAM96671;
At-ECM4a, NP_199315; Zm-GSTL, CAA41447; Os-GSTL, AAF70831; At-GSTL1, AL162973;
Ta-GSTL, CAA76758; hGSTO1-1, NP_004823; hGSTO2-2; NP_899062; So-DHAR, AAG24945;
At-DHAR1, AAF98403; BphK, YP_556402; Sp-GSTB, 1F2ED; Pm-GSTB, 2PMTA; Ec-GSTB,
1A0F_A; PcpC, AAM96673 Os, Oryza sativa; Pm, Proteus mirabilis; So, Spinacia oleracea; Ta,
Triticum aestivum; Zm, Zea mays.
and an N-terminal cysteine residue plays the catalytic role in
Beta, Omega, Lambda and DHAR classes [23]. PcpF contains a
catalytic cysteine residue at its N-terminus [11]. Therefore, PcpF
and representative GSTs from each class containing the active
cysteine residue were analysed by multi-sequence alignment, and
the relationship was presented in a phylogenetic tree (Figure 2).
The PcpF homologues selected for alignment were Re-YqjG and
Ec-YqjG of bacteria, At-ECM4a (A. thaliana ECM4a) of plant
and ECM4 of yeast. PcpF and homologues formed a distinct
group, more related to the Omega, Lambda and DHAR classes
than to the Beta class. PcpC was also included in the analysis
because it catalyses GSH-dependent dechlorination of TeCH [7]
and an N-terminal cysteine residue is involved in the catalysis
[10]. PcpC has been characterized as a Zeta class GST [26].
Substrate specificities of PcpF and related GSTs
PcpF reduces GS-TriCH and GS-DiCH to TriCH and DiCH
with GSH as a co-substrate; however, little is known about
PcpF as a GST [11]. The most highly related GST that has
been characterized is ECM4, assigned to the Omega class [12].
Thus the substrates of Omega class GSTs were tested with PcpF.
For comparison, ECM4, GSTO1-1, GSTO2-2 and two bacterial
PcpF homologues (Ec-YqjG and Re-YqjG) were tested. Among
the substrates tested (Table 1), the human GSTs did not use
GS-TriCH as a substrate, whereas the other enzymes did. In
Table 1 Activities of recombinant GSTs from various species
◦
Specific activities are reported as substrate consumed (μmol · min−1 · mg−1) of protein at 23 1 C and are means S.D. Assays were performed with 5 mM GSH except for the ethacrynic acid
+
+
−
−
assay which used 1 mM GSH. ND, no apparent activity was detected; DiCPGS, S-(2ꢁ4ꢁ-dichlorophenacyl)glutathione.
Enzyme substrate
PcpF
PcpF C53S
Re-YqjG
Ec-YqjG
ECM4
ND
hGSTO1-1
hGSTO2-2
+
+
CDNB
ND
0.09 0.01
2.66 0.12
23.1 0.8
2.49 0.31
1.91 0.14
0.22 0.01
ND
0.11 0.01
ND
ND
0.26 0.05
1.67 0.13
37.9 0.8
2.28 0.13
1.71 0.16
ND
0.07 0.01
1.76 0.07
9.54 0.23
1.30 0.24
0.27 0.03
0.02 0.01
0.01 0.01
−
−
+
+
+
+
+
+
+
Ethacrynic acid
GS-TriCH
HED
0.13 0.01
0.02 0.01
ND
0.37 0.06
ND
−
−
−
−
−
−
−
+
+
+
+
3.18 0.06
−
−
−
−
+
+
+
+
+
+
ND
ND
ND
ND
22.9 0.3
3.91 0.09
9.73 0.08
−
−
−
−
−
−
DMAV
DHA
1.42 0.17
0.074 0.008
0.46 0.02
+
+
+
+
+
+
−
−
−
−
−
−
+
+
+
+
+
+
0.88 0.02
ND
0.57 0.01
26.5 0.53
−
−
−
−
−
−
+
+
DiCPGS
ND
ND
1.56 0.01
ND
−
−
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Figure 3 Alignment of peptide sequences of PcpF homologues with hGSTO1-1
®
GenBank accession numbers for the indicated protein sequences are given Figure 2. Underlined amino acid residues are involved in GSH-binding, and bold residues are involved in the binding of
the second substrate. The secondary structures (α-helixes and β-sheets) of hGSTTO1-1 are shown for reference.
addition, only the human GSTs used CDNB as a substrate, but the
activity was very weak. In comparison, Proteus mirabilis GSTB1–
1 (Pm-GSTB) has a specific activity of 1.1 μmol · min−1 · mg−1
of protein [27], which is 50 times higher than that of GSTO1-
1 (Table 1). All the enzymes reduced the disulfide bond (thiol
transfer), DHA and DMAV with good activities. Most of them had
highest activity as a thiol transferase, using HED as the substrate,
except for GSTO2-2, which had the highest activity for DHA
reduction. Furthermore, all the enzymes had very low activity
for transferring GSH to ethacrynic acid. S-(Phenacyl)glutathiones
are specific substrates of GSTO1-1; hGST2-2 does not use them
[18]. Among the PcpF homologues, only PcpF used S-(2ꢁ,4ꢁ-
dichlorophenacyl)glutathione. Thus S-(phenacyl)glutathiones are
not specific substrates for either the Omega class or PcpF
homologues. A mutant PcpF-C53A, cannot reduce GS-TriCH
[11]. We further tested PcpF-C53A with the substrates listed in
Table 1. The mutant protein showed a similar activity compared
with PcpF for transferring GSH to ethacrynic acid, but lost the
activities towards all the other tested substrates.
Kinetic analysis of PcpF
The GS-TriCH reduction mechanism was also analysed with
+
+
PcpF. The Km values were 4.4 0.6 μM for GS-TriCH and 1.3
−
−
−1
0.4 mM for GSH with a Vmax of 3.4 0.4 μmol · min · mg−1 of
+
−
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L. Xun and others
Figure 4 Double reciprocal plots of initial velocities of PcpF against substrate concentrations
A GS-TriCH enzymatic assay was performed [a coupled assay of PcpF (23 μg · ml−1 at 2.66 μmol · min−1 · mg−1) and glutathione reductase (2.7 μg · ml−1 at 168 μmol · min−1·mg−1)]. Results
are means of two measurements. (A) Enzyme activity was plotted against various concentrations of GSH with four sets of constant concentrations of GS-TriCH. (B) Enzyme activity was plotted against
various concentrations of GS-TriCH with four sets of constant concentrations of GSH. (C) Scheme of deduced Ping Pong mechanism of PcpF from kinetic analysis. The GSH in the scheme can be
replaced by DTT, L-cysteine and 2-mercaptoethanol.
Table 2 Kinetic parameters with different thiols
Native forms of GSTs
GS-TriCH reduction was measured with a spectrometric assay using different small thiols.
2-MerC, 2-mercaptoethanol.
Most GSTs are dimers, including Omega class GSTs; DHAR
and Lambda classes are both monomers [23]. We used both
size-exclusion chromatography and a light-scattering detector
to determine the oligomeric nature of the native forms of
PcpF and ECM4. The retention times of the size-exclusion
chromatography were 17.84 min for PcpF and 17.49 min for
ECM4, correlating to estimated native molecular masses of 65 and
70 kDa. Static light-scattering data estimated the corresponding
molecular masses as 79.7 and 101.2 kDa for PcpF and ECM4
respectively. The calculated dimers of PcpF and ECM4 were
73.8 kDa and 89.6 kDa. The results from both methods suggested
that the enzymes are dimers.
Substrate
kcat
Km (mM)
kcat/Km (s−1 · M−1
)
+
+
DTT
GSH
2-MerC
L-Cysteine
0.89 0.02
2.08 0.25
0.26 0.03
1.3 0.4
3430
1600
1172
187
−
−
+
+
−
−
+
+
1.09 0.06
0.93 0.15
−
−
+
+
0.37 0.09
1.97 0.15
−
−
protein. Given the high Km values for GSH, we increased GSH
from 1 mM to 5 mM in most assays when the specific activities
were determined (Table 1). However, for the ethacrynic acid assay,
1 mM GSH was used because of the spontaneous reaction between
GSH and ethacrynic acid being too high at 5 mM GSH. Kinetic
analysis with various concentrations of GSH and GS-TriCH
produced parallel lines in double reciprocal plots (Figures 4A
and 4B), suggesting that PcpF catalyses GS-TriCH reduction via
a Ping Pong mechanism. We further tested whether PcpF could
use other small thiols for GS-TriCH reduction. Besides GSH,
PcpF used L-cysteine, 2-mercaptoethanol and DTT to reduce
GS-TriCH. The formation of TriCH was confirmed by HPLC
analysis. The end-products were expected to be GS–S-DTT, GS–
SG, GS–S-CH3-CH2OH, or GS–S-Cys, depending on the small
thiol used. As glutathione reductase is known to effectively reduce
asymmetric substrate analogues, including GS–S-nitrobenzoate
and GS–S-nitropyridine [28] as well as GS–S-CH3 [29], we
applied glutathione reductase to reduce the mixed disulfide bond.
The consumption of NADPH was observed and used to determine
the kinetic parameters (Table 2). As judged by kcat/Km values,
PcpF used the substrates in descending order: DTT, GSH, 2-
mercaptoethanol and L-cysteine; however, GSH supported the
fastest catalytic turnover.
DISCUSSION
PcpF and homologues belong to a new class of GSTs
Phylogenetic analysis grouped PcpF homologues into a distinct
class that is closely related to the Omega, Lambda and DHAR
classes (Figure 2); they are more related to the Omega class
because both have the typical dimeric forms of GSTs, and the
Lambda and DHAR classes are monomers [23]. The four groups
of GSTs all have poor or no activities for transferring GSH to
standard GST substrates, e.g. CDNB (Table 1), but are able to
catalyse GSH-dependent reductions. The Lambda class GSTs
can only catalyse thiol transfer (disulfide bond reduction) and
the DHAR class is known to perform both thiol transfer and DHA
reduction [23]. The limited substrate range may be attributable
to the monomeric nature of DHAR and Lambda class GSTs
because the dimer interface of GSTs is involved in substrate
binding [22]. The Omega class GSTs catalyse additional
reactions, including DMAV reduction [21]. The PcpF homologues
can further reduce GS-TriCH (Table 1). Thus both phylogenetic
and biochemical evidence separates PcpF homologues from the
Omega class GSTs. ECM4 is grouped with PcpF homologues,
although it only has 15.7% sequence identity with hGSTO1-1.
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425
Figure 5 Proposed SN2 reaction between PcpF-Cys53 thiolate and GS-TriCH
The Cys53 thiolate, created by its location next to α-helix-1, attacks the sulfur atom of GS-TriCH, which should be slightly electron-deficient due to the strong electron-withdrawing effect of the
aromatic TriCH. The result is the formation of PcpF-Cys53-S–SG, releasing TriCH. The mixed disulfide bond (PcpF-Cys53-S–SG) is reduced by small thiols, including GSH, DTT, 2-mercaptoethanol
and L-cysteine.
It is generally accepted that GSTs with less than 30% sequence
the reaction with a catalytic efficiency (kcat/Km) approx. 10-
fold that when using L-cysteine. hGSTO1-1 catalysed a similar
reaction by using GSH to reduce S-(phenacyl)glutathiones, and
2-mercaptoethanol can be used to replace GSH for the reaction
[18].
identity are assigned to separate classes [22].
ECM4 is a well-known protein among PcpF homologues
Among the PcpF-related proteins, yeast ECM4 is the best
characterized. ECM4 is a protein involved in cell-surface
biosynthesis and architecture. ECM4 was identified as a
transposon mutant hypersensitive to Calcofluor White (a
negatively charged fluorescent dye with a molecular mass of
960.9 kDa) that binds to nascent chains of chitin, a component
of yeast cell walls. S. cerevisiae ecm4 mutants show increased
amounts of chitin, as determined by the N-acetylglucosamine
contents [30]. The expression of the ECM4 gene is up-
regulated when yeast is exposed to genotoxic agents, such as
methyl methanesulfonate, cisplatin and bleomycin [31]. Methyl
methanesulfonate alkylates guanine to form 7-methylguanine
adducts, cisplatin is a DNA cross-linking agent and bleomycin
induces oxidative damage to DNA. It is unclear how ECM4 is
involved in cell-wall synthesis and in protection against genotoxic
agents. ECM4 has recently been characterized as an Omega class
GST (GTO2) for its ability to use the same substrates of the
Omega class GSTs [12]. In the present study we regroup ECM4
with PcpF homologues, into a new class of GSTs, on the basis
of phylogenetic analysis (Figure 2) and their abilities to reduce
GS-TriCH (Table 1). The establishment of a new GST class is in
agreement with the conserved domain database that has an ECM4
group, COG0435, which is not assigned to the Omega class GSTs
[13]. The four PcpF homologues in A. thaliana have all been
identified as ECM4-like proteins. The listed representatives of
COG0435 (ECM4 group) contain both ECM4 and Ec-YqjG.
Proposed role of Cys53 and Pro54 in catalysis
The phylogenetic tree (Figure 2) is in agreement with the active-
site motifs, which also grouped PcpF and homologues separately
from the Beta class. The conserved N-terminal active-site motif of
the Beta class is Cys-Ser, the cysteine residue of which is involved
in interacting with the thiol group of the bound GSH [32]. PcpF
homologues, and the Omega, Lambda and DHAR class GSTs, all
have an N-terminal active-site motif of Cys-Pro. The two residues,
Cys32 and Pro33 of hGSTO1-1, are located at the start of α-helix
1 (Figure 3), and the proximity of Cys32 to the positive end of
the α-helix dipole promotes the formation of Cys32 thiolate [21].
The Cys-Pro residues are Cys53 and Pro54 in PcpF (Figure 3).
The Cys53 thiolate should be a strong nucleophile, and the sulfur
atom of GS-TriCH should be slightly electron-deficient due to the
strong electron-withdrawing effect of the aromatic ring of TriCH
(Figure 5). Thus a typical bimolecular nucleophilic substitution
(SN2 reaction) takes place at the active site of PcpF: the thiolate
attacks the sulfur atom, forming PcpF-Cys53-S–SG and expelling
TriCH. The apparent difference in key amino acid residues for
substrate binding (Figure 3) may prevent the two human Omega
class GSTs from binding GS-TriCH.
Physiological roles of GS-TriCH reductases
A potential role as thiol transferase for PcpF and homologues
is uncertain as the activities with HED, although significant
(Table 1), are much lower than the activity of glutaredoxin 1 of the
alga Chlamydomonas (Vmax = 710 μmol · min−1·mg−1) [33]. PcpF
homologues all have good DHAR activities (Table 1), but are
much lower than that (at 936 μmol · min−1 · mg−1) of At-DHAR1
[23]. Among the activities catalysed by PcpF homologues,
only GS-TriCH reduction is unique to the group (Table 1).
Thus PcpF homologues are tentatively called S-glutathionyl-
(chloro)hydroquinone reductases. PcpF was previously referred
as a glutathionyl-chlorohydroquinone lyase for its ability for
reducing GS-TriCH and GS-DiCH conjugates [11]. As PcpF and
homologues catalyse GSH-dependent reduction of disulfide bond
(thiol transfer), DHA, DMAV and GS-TriCH, naming them as
Reaction mechanism
Combining the kinetic data (Figure 4) and the catalytic role of
Cys53 of PcpF [11], a Ping Pong reaction mechanism for GS-
TriCH reduction is proposed (Figure 4C). First, PcpF reacts with
GS-TriCH to release TriCH and to form a disulfide bond of
PcpF-Cys53-S–SG. Then, a GSH molecule comes in to regenerate
PcpF-Cys53-SH with the concomitant formation of GS–SG. DTT,
2-mercaptoethanol (HS-CH3-CH2OH) and L-cysteine also react
with PcpF-Cys53-S–SG to regenerate PcpF-Cys53-SH, with the
commitment production of GS–S-DTT, GS-S-CH3-CH2OH and
GS-S-Cys respectively. As DTT and 2-mercaptoethanol are not
present in the cytoplasm, GSH is the natural substrate for
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L. Xun and others
4
5
Cai, M. and Xun, L. (2002) Organization and regulation of pentachlorophenol-degrading
genes in Sphingobium chlorophenolicum ATCC 39723. J. Bacteriol. 184, 4672–4680
Dai, M., Rogers, J. B., Warner, J. R. and Copley, S. D. (2003) A previously unrecognized
step in pentachlorophenol degradation in Sphingobium chlorophenolicum is catalyzed by
tetrachlorobenzoquinone reductase (PcpD). J. Bacteriol. 185, 302–310
Xun, L. and Orser, C. S. (1991) Purification and properties of pentachlorophenol
hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723. J. Bacteriol.
173, 4447–4453
Orser, C. S., Dutton, J., Lange, C., Jablonski, P., Xun, L. and Hargis, M. (1993)
Characterization of a Flavobacterium glutathione S-transferase gene involved in reductive
dechlorination. J. Bacteriol. 175, 2640–2644
Xun, L., Topp, E. and Orser, C. S. (1992) Purification and characterization of a
tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp. J.
Bacteriol. 174, 8003–8007
reductases, instead of lyases, is more appropriate. The proximity
of pcpC and pcpF genes on chromosome of S. chlorophenolicum
ATCC 29723 has led to the identification of the role of PcpF in
PCP degradation. However, the activity for GS-TriCH reduction
is not limited to PcpF. All of the tested homologues reduced GS-
TriCH at similar rates (Table 1). The wide presence of PcpF
homologues in bacteria, archaea, fungi and plants suggests a
physiological role beyond the degradation of PCP, a chemical only
introduced into the environment recently [2]. As S-glutathionyl-
(chloro)hydroquinone reductases are common, but GS-TriCH is
not, it may not be far-fetched to hypothesize that S-glutathionyl-
(chloro)hydroquinone reductases can use other substituted
GS-hydroquinones as their substrates. Both o- and p-substituted
benzoquinones can react with GSH to form substituted GS-
hydroquinones, and the reaction can be spontaneous or catalysed
by GSTs [34,35]. The physiological advantage of reducing
some GS-hydroquinones needs further investigation; however, the
maintenance role of PcpF in PCP degradation is clearly beneficial
to S. chlorophenolicum [11].
6
7
8
9
Xun, L., Bohuslavek, J. and Cai, M. (1999) Characterization of
2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA) of Sphingomonas
chlorophenolica ATCC 39723. Biochem. Biophys. Res. Comm. 266, 322–325
10 McCarthy, D. L., Navarrete, S., Willett, W. S., Babbitt, P. C. and Copley, S. D. (1996)
Exploration of the relationship between tetrachlorohydroquinone dehalogenase and the
glutathione S-transferase superfamily. Biochemistry 35, 14634–14642
11 Huang, Y., Xun, R., Chen, G. and Xun, L. (2008) Maintenance role of a
glutathionyl-hydroquinone lyase (PcpF) in pentachlorophenol degradation by
Sphingobium chlorophenolicum ATCC 39723. J. Bacteriol. 190, 7595–7600
12 Garcera, A., Barreto, L., Piedrafita, L., Tamarit, J. and Herrero, E. (2006) Saccharomyces
cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol
transferases. Biochem. J. 398, 187–196
13 Tatusov, R. L., Fedorova, N. D., Jackson, J. D., Jacobs, A. R., Kiryutin, B., Koonin, E. V.,
Krylov, D. M., Mazumder, R., Mekhedov, S. L., Nikolskaya, A. N. et al. (2003) The COG
database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41
14 Schmuck, E. M., Board, P. G., Whitbread, A. K., Tetlow, N., Cavanaugh, J. A., Blackburn,
A. C. and Masoumi, A. (2005) Characterization of the monomethylarsonate reductase and
dehydroascorbate reductase activities of Omega class glutathione transferase variants:
implications for arsenic metabolism and the age-at-onset of Alzheimer’s and Parkinson’s
diseases. Pharmacogenet. Genomics 15, 493–501
15 Habig, W. H., Pabst, M. J. and Jakoby, W. B. (1974) Glutathione S-transferases: the first
enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139
16 Holmgren, A. and Aslund, F. (1995) Glutaredoxin. Methods Enzymol. 252, 283–292
17 Whitbread, A. K., Masoumi, A., Tetlow, N., Schmuck, E., Coggan, M. and Board, P. G.
(2005) Characterization of the omega class of glutathione transferases. Methods Enzymol.
401, 78–99
18 Board, P. G. and Anders, M. W. (2007) Glutathione transferase omega 1 catalyzes the
reduction of S-(phenacyl)glutathiones to acetophenones. Chem. Res. Toxicol. 20,
149–154
Evolution
GSTs share strong structure similarity despite the low sequence
identity (for a review, see [22]). The GST fold consists of
an N-terminal glutaredoxin-fold domain, a C-terminal α-helical
domain and a cleft between the two domains that houses
the active site. The N-terminal domain contains most of the
residues for GSH binding, whereas the C-terminal domain is
usually responsible for the binding of the second substrate. The
glutaredoxin-fold belongs to the thioredoxin-fold superfamily.
It has been suggested that conjugating GSTs are evolved from
oxidoreductases with GSH as the reductant [36]. Apparently,
S-glutathionyl-(chloro)hydroquinone reductases, DHAR, and
Omega class and Lambda class GSTs have weak or no conjugation
abilities, and all have the thiol transferase activity of glutaredoxin
[16]. They may represent a branch of GSTs that catalyse
mainly GSH-dependent oxidoreduction (Table 1), rather than
conjugation.
19 Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam,
H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R. et al. (2007) Clustal W and Clustal X
version 2.0. Bioinformatics 23, 2947–2948
20 Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular evolutionary
genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599
21 Board, P. G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L. S., Schulte, G. K.,
Danley, D. E., Hoth, L. R., Griffor, M. C., Kamath, A. V. et al. (2000) Identification,
characterization, and crystal structure of the Omega class glutathione transferases. J. Biol.
Chem. 275, 24798–24806
22 Sheehan, D., Meade, G., Foley, V. M. and Dowd, C. A. (2001) Structure, function and
evolution of glutathione transferases: implications for classification of non-mammalian
members of an ancient enzyme superfamily. Biochem. J. 360, 1–16
AUTHOR CONTRIBUTION
Luying Xun conceived and co-ordinated the study. He also conducted the majority of
the experiments together with Sara Belchik, Randy Xun and Yan Huang. Huina Zhou
and Philip Board generated and purified hGSTO2-2 protein and synthesized S-(2ꢁ,4ꢁ-
dichlorophenacyl)glutathione. Emiliano Sanchez and ChulHee Kang determined the native
molecular masses of PcpF and ECM4. All authors contributed to the writing and pre-
acceptance editing of the manuscript.
23 Dixon, D. P., Davis, B. G. and Edwards, R. (2002) Functional divergence in the glutathione
transferase superfamily in plants: identification of two classes with putative functions in
redox homeostasis in Arabidopsis thaliana. J. Biol. Chem. 277, 30859–30869
24 Board, P. G., Baker, R. T., Chelvanayagam, G. and Jermiin, L. S. (1997) Zeta, a novel class
of glutathione transferases in a range of species from plants to humans. Biochem. J. 328,
929–935
25 Li, J., Xia, Z. and Ding, J. (2005) Thioredoxin-like domain of human Kappa class
glutathione transferase reveals sequence homology and structure similarity to the Theta
class enzyme. Protein Sci. 14, 2361–2369
FUNDING
The research was supported by the U.S.A. National Science Foundation [grant number
MCB-0323167]; and by the Australian National Health and Medical Research Council.
S.M.B. was a fellowship recipient of an National Institutes of Health Biotechnology Training
Grant.
26 Anandarajah, K., Kiefer, P. M. J., Donohoe, B. S. and Copley, S. D. (2000) Recruitment of
a double bond isomerase to serve as a reductive dehalogenase during biodegradation
of pentachlorophenol. Biochemistry 39, 5303–5311
27 Casalone, E., Allocati, N., Ceccarelli, I., Masulli, M., Rossjohn, J., Parker, M. W. and Di
Ilio, C. (1998) Site-directed mutagenesis of the Proteus mirabilis glutathione transferase
B1-1 G-site. FEBS Lett. 423, 122–124
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Received 8 December 2009/3 March 2010; accepted 13 April 2010
Published as BJ Immediate Publication 13 April 2010, doi:10.1042/BJ20091863
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