(Received for publication, July 13, 1995; and in revised form, August 10, 1995)
From the
Three different molecular masses (24, 22, and 20 kDa) of
antioxidant proteins were purified in Escherichia coli. These
proteins exhibited the preventive effects against the inactivation of
glutamine synthetase activity and the cleavage of DNA by a
metal-catalyzed oxidation system capable of generating reactive oxygen
species. Their antioxidant activities were supported by a
thiol-reducing equivalent such as dithiothreitol. Analysis of the
amino-terminal amino acid sequences and the immunoblots between 24- and
22-kDa proteins indicates that the 24-kDa protein is an intact form of
the 22-kDa protein that was previously identified 22-kDa subunit (AhpC)
of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We
isolated and sequenced an E. coli genomic DNA fragment that
encodes 20-kDa protein. Comparison of the deduced amino acid sequence
of the 20-kDa protein with that of AhpC revealed no sequence homology.
A search of a data bank showed that the 20-kDa protein is a new type of
antioxidant enzyme. The synthesis of this novel 20-kDa protein was
increased in response to oxygen stress during growth. The 20-kDa
protein resides mainly in the periplasmic space of E. coli,
whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa
protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This
20-kDa protein is thus named ``thiol peroxidase,'' which
could act as an antioxidant enzyme removing peroxides or
HO
within the catalase- and
peroxidase-deficient periplasmic space of E. coli.
In an aerobic environment, reactive oxygen species
(O, H
O
, ROOH, and
HO
) are generated by many physiological processes such
as incomplete reduction of molecular oxygen during respiration, NADPH
oxidation linked to respiratory burst during phagocytosis, and redox
cycling of xenobiotics(1) . To prevent the deleterious effect
of oxygen species, cells have equipped with a number of antioxidant
enzymes, including catalases, peroxidases, and superoxide dismutase.
Recently, a 25-kDa antioxidant enzyme was purified from various
eukaryotes, including yeast(2) , human erythrocyte(3) ,
and rat brain (4) . These enzymes prevent the oxidative damage
induced by oxidation system capable of generating reactive oxygen
species in the presence of a thiol reducing equivalent such as
DTT()(2, 3, 4) . However, such an
antioxidant activity was abolished without a thiol-reducing equivalent.
Thus, this enzyme has been named ``TSA''
(``thiol-specific antioxidant protein''). Previously, we have
reported that the yeast TSA has a capability to destroy
H
O
in the presence of DTT (5) , and
such a peroxidase activity was greatly enhanced by the in vivo thiol-regenerating system (thioredoxin-thioredoxin
reductase-NADPH)(6) . However, its physiological significance
as a peroxidase is still debatable because of the existence of
conventional catalases and peroxidases in eukaryotic cytoplasm. Yeast
and human genes that encode the 25-kDa TSA have been cloned and
sequenced(7, 8) . The deduced amino acid sequences
showed no homology to known antioxidant
enzymes(8, 9) . An analysis of data bases revealed 27
additional protein sequences showing homology to the 25-kDa TSA. The
biochemical functions of these homologous proteins (TSA family) are not
yet clarified except for AhpC, one subunit of alkyl hydroperoxide
reductase found in Salmonella typhimurium and Escherichia
coli(10) . The alignment of amino acid sequences of TSA
family revealed two highly conserved cysteine residues. The yeast TSA
whose conserved cysteines were replaced with serines completely lost
antioxidant activity, which indicates that these cysteine residues are
essential for the activity(11, 12) . Thus, the
TSA/AhpC family has been suggested to be a new type of peroxidase
containing functional cysteines(6, 9, 12) .
During aerobic growth, E. coli can be exposed to endogenous
and exogenous reactive oxygen species from various oxidation reactions.
These reactive oxygen species are known to damage cellular
constituents. Alkyl hydroperoxides among many products of oxygen
radical damage have a capability to initiate and propagate free radical
chain reactions leading to DNA and membrane damages(13) . In
eukaryotes, glutathione peroxidases catalyze the reduction of alkyl
hydroperoxides to the corresponding alcohols and HO.
However, there has been no evidence that glutathione peroxidase exists
in prokaryotes. A peroxidase was identified in both Salmonella
typhimurium and E. coli(10) . The purified
activity required two separable subunits, 22- and 57-kDa proteins. The
57-kDa AhpF-linked and 22-kDa AhpC proteins converts alkyl
hydroperoxides to the corresponding alcohols. This enzyme (AhpF/AhpC),
hence, was suggested to serve as a prokaryotic equivalent to the
glutathione reductase/glutathione peroxidase system in eukaryotes.
From E. coli, we purified three proteins (20, 22, and 24 kDa) showing thiol-dependent antioxidant activities. The 20-kDa antioxidant enzyme among the three proteins was shown to be a novel antioxidant enzyme, which resided in the periplasmic space of E. coli. In this paper we reported the purification and characterization of a novel E. coli 20-kDa antioxidant protein showing peroxidase activity and discussed its physiological function in the periplasmic space on the basis of thioredoxin (Trx)-linked ``thiol peroxidase.''
Figure 1:
SDS-PAGE analysis of p20 and p22 at
different stages of purification. Protein samples from different stages
of purifications were electrophoresed in 12% SDS-PAGE gel. Lane A1 contains 80-µg sample derived from a DEAE column. Lane
A2, 40-µg sample from peak II of the first G-75 column. Lane A3, 20-µg sample from phenyl-Sepharose CL-4B column. Lane A4, 10 µg from the second G-75 column. Lane
A5, 5 µg from the Sephadex G-50 column. Lane A6, 2.5
µg from Acell-QMA column. Lanes A1-A6 are samples
from purification steps for p20 (20-kDa protein). Lanes A10,
A9, and A8 are samples derived from purification stages
for p22: lane A10, 40-µg sample from peak I of first
Sephadex G-75 column. Lane A9, 20-µg sample from
phenyl-Sepharose CL-4B. Lane A8, 5-µg sample from the
G-100 column. Lanes B1 and B2 are 2.5 µg of p22
and p20, respectively. Lanes A7 and B3 are size
markers. The molecular masses, from the bottom, are 14.4, 21.5, 31, 45,
66.2, and 97.4 kDa. All of samples except for lanes B4 and B5 were reduced and denatured in SDS--mercaptoethanol
sample buffer. In case of lanes B4 and B5, 2.5 µg
of p20 or p22 was denaturated without
-mercaptoethanol,
respectively. The arrow shown on lane B5 indicates
the dimer form of p22.
The G-75 gel permeation chromatography yielded two
peaks showing thiol-dependent antioxidant activity, peaks I and II (not
shown). From the later peak II, p20 was purified to homogeneity by four
sequential chromatographic steps on phenyl, G-75, G-50, and Accell-QMA
(Waters) chromatographies. From the peak I, p22 was homogeneously
obtained from two additional purification steps using phenyl column and
G-75 columns (Fig. 1A). In the nonreducing gel without
-mercaptoethanol, p22 was detected at the molecular size
corresponding to the dimer form, suggesting an intermolecular disulfide
bond (lanes 1 and 5 in Fig. 1B),
whereas p20 at the molecular size of the monomer (lanes 2 and 4 in Fig. 1B).
From the immunoblot of the crude extract of E. coli with the p22-specific polyclonal antibodies, p24 was detected as a unique band (not shown). The immunoblot analysis of each purification step showed that during further purifications the p24 band disappeared, whereas the p22 band appeared. To conform p24 as a native form of p22, we tried to purify p24. From the ammonium sulfate fractions (30-60%) of the crude extract, the p24 was purified to homogeneity by three sequential chromatographic steps on Sephadex G-75, phenyl-Sepharose CL-4B, and Sephacryl S-200 HR chromatographies. The reducing SDS-PAGE analysis for leading fractions of the S-200 showed that the p24 is approximately near to the upper exclusion limit of S-200 (250-kDa). This suggests that p24 may exist as an aggregated form. Such an aggregated property was also reported in the case of 25-kDa TSA from yeast(2) . In contrast to p24, the elution profile of p20 from the S-200 column indicates that p20 exists in the monomer form having molecular mass of 20 kDa. The native molecular mass of p20 was estimated to be 16.8 kDa on the comparison of the elution volume of p20 from Sephacryl S-200 gel filtration chromatography with those of protein standards (see ``Experimental Procedures'').
Figure 2: Nucleotide sequence and deduced amino acid sequence of the p20 gene. Nucleotides are numbered (left margin) beginning with the first base of the ATG initiator codon. Deduced amino acid residues are numbered (left margin, in parentheses) beginning with the serine immediately after the initiating methionine. The two cysteine residues (italic character) are underlined. The regions corresponding to the determined amino-terminal amino acid sequence and the sequences of three tryptic TNB-conjugated peptides (C1, C3, and C4) are underlined. This sequence has GeneBank(TM) accession number U33213.
The gene encoding p20 was cloned as a 2.2-kilobase fragment and was sequnced. An open reading frame was identified and found to encode a polypeptide of 167 amino acids with a calculated molecular mass of 17,764 daltons (Fig. 2). The amino acid sequence of p20 contains two cysteine residues. The amino acid sequence of p20 does not shows a significant similarity to those of TSA/AhpC family and conventional antioxidant enzymes. The number of cysteines in p20 was also determined by the DTNB titration method after the protein was reduced with 1 mM DTT in the presence of 6 M guanidine chloride. Approximately two sulfhydryl groups per 20-kDa polypeptide were detected. It has been reported that two cysteines exist in the TSA/AhpC family proteins. The amino acid sequences of the regions containing the cysteines (VCP1: FTFVCPTE and VCP2: GEVCPA) are perfectly conserved(8, 12) . The amino acid sequence of the region containing two cysteines in p20 shows no significant homology to any amino acid sequence of the VCP1 and VCP2 domains. Thus, this result suggests that p20 could not be a member of the TSA/AhpC family.
Figure 3:
Protection of glutamine synthetase by p20
and p24 against the DTT/Fe (thiol MCO) system. The
inactivation mixture contained 10 µg of E. coli glutamine
synthetase, 10 mM DTT for the thiol MCO system or 10 mM ascorbate for the non-thiol MCO system, 3 µM
FeCl
, 50 mM Hepes, pH 7.0, in a total volume of
100 µl. All reactions were carried out at 37 °C. At indicated
times, aliquots (10 µl) were removed and assayed for glutamine
synthetase activity. Each inactivation reaction mixture contained as
follows: curve 1 in A and B, glutamine
synthetase plus 1 mM EDTA; curve 2 in A and B, thiol MFO system plus 50 nM p20 or 50 nM p24, respectively; curves 3 in A and B,
non-thiol MCO system plus 50 nM p20 or p24, respectively. Curves 4 and 5 in A and B represent
the inactivation of glutamine synthetase by non-thiol (ascorbate) MFO
system (curve 4) or by thiol MCO system (curve 5)
without p20 (A) and p24 (B).
p20 was examined for glutathione peroxidase under the assay conditions described under ``Experimental Procedures,'' because p20 was the required thiol-reducing equivalent such as DTT to maintain its antioxidant activity. Ten µg of p20 did not cause significant oxidation of NADPH. Two mg of p20 (100 nmol) was applied to a double beam atomic adsorption spectrophotometer (GBC 902) for the determination of selenocysteine in p20. However, a significant amount of selenium was not detected (not shown). These results indicate that p20 is not a type of selenium-dependent glutathione peroxidase.
When thiol is replaced
with another electron donor (e.g. ascorbate), p20 no longer
protects against MCO system-induced glutamine synthetase inactivation. Fig. 4shows that the antioxidant activity of p20 becomes
restored, showing a saturation tendency as the concentration of DTT or
GSH in the non-thiol-MCO system (e.g. ascorbate/Fe/O
) was increased. These
results support the possibility that a thiol-reducing equivalent such
as DTT or GSH could give p20 potential for preventing the oxidative
damage induced by the ascorbate MCO system. To examine this, p20 was
reacted with N-ethylmaleimide, a cysteine-specific
modification reagent with or without DTT. p20 was preincubated with 1
mM DTT and then reacted with 5 mMN-ethylmaleimide. The resulting p20 did not prevent the
inactivation of glutamine synthetase activity and the cleavage of
plasmid DNA by thiol MCO system (not shown). The reaction of p20 with N-ethylmaleimide without DTT did not result in the
inactivation of the antioxidant activity (not shown). These results
indicate that a functional sulfhydryl group(s) (i.e. cysteine
residue) of p20 is involved in the antioxidation reaction, and the
resulting intramolecular disulfide bond can be regenerated by a
thiol-reducing equivalent such as DTT.
Figure 4: The concentration-dependent effects of DTT and glutathione (GSH) on the preventive activity of p20 against the inactivation of glutamine synthetase by ascorbate MFO system. A, various amounts of DTT were added into the ascorbate (i.e. non-thiol) MCO system containing 100 nM p20. After 40 min at 37 °C, the remaining glutamine synthetase activities were measured. B, various amounts of GSH were added into the non-thiol MFO system containing 100 nM p20. Lines 2 in A and B are the corresponding control experiments without p20.
An enzymatic thiol regenerating system (Trx/Trx reductase/NADPH) was tested for its ability to regenerate the activity of p20. This system gave p20 capability for protecting the inactivation of glutamine synthetase by ascorbate MCO system. Fig. 5, A and B, show the NADPH dependence of the antioxidant activity of p20 against ascorbate MCO system in the presence of Trx and Trx reductase. As the concentration of NADPH was increased, the antioxidant activity of p20 became restored showing saturation pattern. The potency of the Trx system containing saturation concentration of NADPH (5 mM) was compared with that of DTT-supported system containing an excess amount of DTT (10 mM) by measuring the ability of p20 to prevent the glutamine synthetase inactivation in the presence of varied concentrations of p20. The concentration of p20 required for 50% protection of glutamine synthetase in the presence of the Trx system was 18 nM and was 75 nM in the presence of DTT (not shown). The higher efficiency of Trx system than that of DTT or GSH suggests that the Trx system, not GSH, is likely to reduce the oxidized p20 in vivo.
Figure 5: Trx-linked antioxidant activity of p20. Glutamine synthetase was subjected to inactivation in 50 µl of a reaction mixture containing the non-thiol (10 mM ascorbate) MCO system, 100 nM p20, 25 µg/ml TrX, 25 µg of Trx reductase, 50 mM Hepes-NaOH (pH 7.0), and various concentrations of NADPH. At various times, 8-µl aliquots were removed and assayed for glutamine synthetase. Curve 1 in A, 5 mM NADPH; curve 2 in A, 1 mM NADPH; curve 3 in A, whole component without p20; curve 4 in A, whole component without Trx. Curve 1 in B, glutamine synthetase activity after 30 min at 37 °C, in whole components containing various concentrations of NADPH ranging from 0.625 to 5 mM. Curve 2 in B, control glutamine synthetase activity in whole components containing varying concentrations of NADPH without p20.
Figure 6:
Removal
of HO
by p20 linked to the Trx system.
Peroxidase reaction was carried out in a 0.5-ml reaction mixture
containing 50 mM Hepes-NaOH (pH 7.0), 50 nM p20, 12.5
µg of Trx, 12.5 µg of Trx reductase, 0.12 mM
H
O
, 0.25 mM NADPH (curve 2)
at 37 °C. Line 1, control experiment the on whole reaction
component minus p20. At the indicated time, a 50-µl sample was
removed, and the concentration of remaining H
O
was measured with the use of ferrithiocyanate as
described(5, 32) .
Figure 7:
Functional tight coupling between
peroxidase activity of p20 and the Trx system. The decrease of NADPH
was spectrophotometrically monitored in a 300-µl reaction mixture
containing the Trx system (0.25 mM NADPH, 12.5 µg of Trx
and 12.5 µg of Trx reductase), 50 mM Hepes-NaOH (pH 7.0),
50 nM p20, and various concentrations of HO
(A) and t-butyl hydroperoxide (C).
After 2-min preincubation at 25 °C, the reaction was started by the
addition of substrate, H
0
, or t-butyl
hydroperoxide to the reaction mixture. A, NADPH oxidation of
Trx system coupled to the consumption of H
O
by
p20. A: traces 0-5, 0.044, 0.059, 0.088, 0.118,
and 0.147 mM H
O
, respectively. B, Lineweaver-Burk plot of the initial rate of NADPH oxidation versus the concentration of H
O
added. C, NADPH oxidation of the Trx system coupled to the
consumption of t-butyl hydroperoxide by p20. C: traces
0-4, 0, 0.06, 0.03, 0.023, and 0.015 mMt-butyl hydroperoxide, respectively. D,
Lineweaver-Burk plot of the initial rate of NADPH oxidation versus the concentration of t-butyl hydroperoxide. One unit of
peroxidase activity represents 1 µmol of peroxide which is
converted to product/µg of p20/min.
The cells grown under anaerobic or aerobic conditions were subjected to osmotic shock. Proteins of periplasmic space were released into solution by the osmotic shocked treatment, whereas enzymes of the matrix space were retained(16) . Catalase, which is known to be a matrix enzyme in E. coli, used as a marker enzyme. On the Western blot with polyclonal antibodies of catalase, the cytoplasmic catalase could not be detected in the periplasmic protein extract, indicating that the procedures used in preparing the shocked fluid caused little release of matrix enzymes. Therefore, the evident p20 band in the shock fluid (not shown) indicates its existence in the periplasmic space. The comparisons of the band intensities on the Western blot of p20 with that of AhpC denote that p20 is much more abundant in the periplasmic space than AhpC.
In an attempt to clarify the existence of p20 in the periplasmic space, we purified the p20 from the osmotic shock fluid. p20 was purified to homogeneity by three sequential chromatographic steps on phenyl-Sepharose CL-4B and two rounds of G-50. However, the catalase activity was not detected in the periplasmic protein extract, indicating no contamination with cytoplasmic proteins. The immunoblot experiments revealed that the monospecific antibodies prepared against p20 are highly specific to the antigen. To determine the concentration of p20 in the periplasmic space of E. coli, the immunoreactivity was measured with from 10 to 40 µg of soluble proteins prepared from cytoplasm and periplasmic space of E. coli grown under aerobic condition. From the standard immunoblots in which the intensity of immunoblot increased with increasing amounts of purified p20 from 12.5 to 400 ng (not shown), the amount of p20 in the periplasmic space was estimated to be between 0.5 and 1.0% of the total periplasmic proteins, whereas the amount of p20 in the cytoplasm was estimated to be between 0.05 and 0.1% of total cytoplasmic proteins. These results confirm the abundant existence of p20 in the periplasmic space of E. coli.
Recently, a family of TSA proteins, more recently referred to as thioredoxin-dependent peroxidases, has been rapidly growing (2, 3, 4, 5, 6, 7, 8, 9) . The similarity among these proteins, including E. coli AhpC, extended over the entire sequence, especially in the domains (VCP1 and VCP2 domains), which contain highly conserved cysteines(8) . Therefore, AhpC has been suggested to be a prokaryotic counterpart of the eukaryotic TSA(12) .
We purified a novel 20-kDa
antioxidant protein (p20) from E. coli. p20 shares the same
catalytic characteristics of TSA/AhpC proteins (i.e. thiol-dependent antioxidant
properties)(2, 3, 4, 5, 6, 7, 8, 9) .
p20 appeared to have a significant peroxidase activity to destroy
HO
and alkyl peroxide such as t-butyl
hydroperoxide. The predicted amino acid sequence of p20 does not show
any significant homology to those of TSA/AhpC family (Fig. 2). A
data bank search reveals that p20 is a novel E. coli protein.
These results suggest p20 is a novel type of thiol-dependent
peroxidase.
In order to understand a reason for the existence of two
types of peroxidases such as p20 linked to the Trx system and AhpC
linked to F52 (reductase component of alkyl hydroperoxide reductase) in E. coli, the differences of their physiological functions were
examined. Their inducibilities of protein synthesis with response to
oxidative stress were nearly same, but their different cellular
compartmentalizations might give a clue to understand the reason. The
distributions of p20 and p24 (AhpC) between the periplasmic space and
cytoplasm of E. coli were different. p20 appears to be
localized mainly in the periplasmic space, whereas AhpC resides mainly
in the matrix of the cells. On the basis of these observations, it
appears that p20, in the periplasmic space, could serve as a peroxidase
to remove exogenous peroxides, while the AhpC, in the cytoplasm of the
cell, acts as a peroxidase against endogenous peroxides. Analogies to
these cellular localizations were reported in the case of superoxide
dismutase, a metalloenzyme found in all organisms(27) . E.
coli has two isoenzyme forms of superoxide dismutase: iron
superoxide dismutase (28) and manganese superoxide
dismutase(29) . The cellular localizations of these isoenzymes
are different. The periplasmic fluid contained 12 units of the
magnesium form and 68 units of the iron form, whereas the
shock-extracted cells (i.e. cytoplasm) contained 846 units of
the manganese superoxide dismutase and 585 units of iron superoxide
dismutase(26) . Therefore, it appears that in the periplasmic
space of the cells, iron superoxide dismutase converts exogenous
O to H
O
. Without
removing the periplasmic H
O
, very destructive
hydroxyl radicals capable of damaging the cell membrane may be
generated by Fe
via Fenton reaction. We tried to
purify any peroxidase and catalase activities from the periplasmic
fluids of E. coli, but these activities were not found in the
periplasmic space. Thus, it is likely that p20, in the periplasmic
space, might be a unique peroxidase to remove the periplasmic peroxides
such as H
O
and alkyl hydroperoxides.
This
new enzyme shares the similar catalytic cycles of TSA protein
(thioredoxin peroxidase), which involves the transfer of reducing
equivalent by redox active disulfhydryls of Trx. However, this proposed
mechanism is different from the previously reported catalytic cycles of
TSA protein (9) in that the intramolecular disulfide linkage
(not intermolecular disulfide bond of TSA proteins) of p20 was involved
in the cycles, which is supported by the observations of the inactive
monomer form of p20 in the absence of DTT (Fig. 1). p20 contains
neither selenocysteine nor prosthetic group such as a heme or a flavin.
Thus, it will be very interesting to investigate how p20 shows a
peroxidase activity. The mechanism of p20 to destroy peroxides might be
analogous to the mechanism proposed for selenocysteine glutathione
peroxidases that have been not found in prokaryotes. The functional
cysteine could be oxidized to an intermediate,
-Cys-S-OH, on the assumption that redox-active
cysteine of p20 gains abnormal strong nucleophilicity comparable with
that of selenocysteine, -Cys-Se-OH, of glutathione
peroxidase. It is likely that the thiol group in the active cysteine
residue of p20 would gain abnormal nucleophilicity by a
microenvironmental effect. The analogy with the thiol exhibiting an
abnormal strong nucleophilicity was reported previously. The ovothiol,
a mercaptoimidazole, is more effective than catalase in destroying
HO
(30) . The capability of ovothiol was
proven to be due to the strong nucleophilicity of the thiol
group(31) .
In conclusion, the novel 20-kDa antioxidant protein (p20), which is localized in the periplasmic space of E. coli, is a peroxidase linked to the Trx system, but its primary structure differs from that of the TSA/AhpC family of thioredoxin-dependent peroxidases. In order to discriminate this type of E. coli peroxidase having functional cysteine from the selenocysteine peroxidase such as glutathione peroxidase, we tentatively named p20 as ``thiol peroxidase.'' The identity of the normal physiological substrate(s) of thiol peroxidase (whether it is an alkyl hydroperoxide of lipid, hydrogen peroxide, hydroxyl radical, or some other cellular components containing oxygen radical) remains to be determined.
To investigate the mechanism of antioxidant action of thiol peroxidase and its physiological function, we are to make a thiol peroxidase deletion mutant and the point-mutated enzyme whose putative active cysteine(s) is changed to other amino acid.