Protein S-thiolation by glutathionylspermidine (Gsp): The role of Escherichia coli Gsp synthetase/amidase in redox regulation

Article abstract of DOI:10.1074/jbc.M110.133363

Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated byH ₂O₂ oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/glutaredoxin null mutants toH₂O₂ support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.

Full text of DOI:10.1074/jbc.M110.133363

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 33, pp. 25345–25353, August 13, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Protein S-Thiolation by Glutathionylspermidine (Gsp)
□
S
THE ROLE OF ESCHERICHIA COLI Gsp SYNTHETASE/AMIDASE IN REDOX REGULATION*
Received for publication, April 13, 2010, and in revised form, June 2, 2010 Published, JBC Papers in Press, June 8, 2010, DOI 10.1074/jbc.M110.133363
Bing-Yu Chiang‡§, Tzu-Chieh Chen‡§, Chien-Hua Pai‡, Chi-Chi Chou¶, Hsuan-He Chenʈ, Tzu-Ping Ko‡,
Wen-Hung Hsu‡§, Chun-Yang Changʈ, Whei-Fen Wuʈ**1, Andrew H.-J. Wang‡§‡‡2, and Chun-Hung Lin‡§‡‡3
From the ‡Institute of Biological Chemistry, ‡‡Genomics Research Center, and ¶National Research Program for Genome Medicine
Core Facilities for Proteomics Research, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan and the §Institute of
Biochemical Sciences, ʈDepartment of Agricultural Chemistry, and **Department of Biochemical Science and Technology, National
Taiwan University, Taipei 10617, Taiwan
Certain bacteria synthesize glutathionylspermidine (Gsp),
from GSH and spermidine. Escherichia coli Gsp synthetase/am-
idase (GspSA) catalyzes both the synthesis and hydrolysis of
Gsp. Prior to the work reported herein, the physiological role(s)
of Gsp or how the two opposing GspSA activities are regulated
had not been elucidated. We report that Gsp-modified proteins
from E. coli contain mixed disulfides of Gsp and protein thiols,
representing a new type of post-translational modification for-
merly undocumented. The level of these proteins is increased by
oxidative stress. We attribute the accumulation of such proteins
to the selective inactivation of GspSA amidase activity. X-ray
crystallography and a chemical modification study indicated
that the catalytic cysteine thiol of the GspSA amidase domain is
transiently inactivated by H2O2 oxidation to sulfenic acid, which
is stabilized by a very short hydrogen bond with a water mole-
cule. We propose a set of reactions that explains how the levels
of Gsp and Gsp S-thiolated proteins are modulated in response
to oxidative stress. The hypersensitivities of GspSA and GspSA/
glutaredoxin null mutants to H2O2 support the idea that GspSA
and glutaredoxin act synergistically to regulate the redox envi-
ronment of E. coli.
(5). Because GSH is abundant in most organisms (often existing
at 1–10 mM), protein S-glutathionylation (GSH S-thiolation) is
considered to be a reversible and universal cellular process. In
E. coli, GSH and spermidine (Spd)4 form N1-glutathionylsper-
midine (Gsp) via an ATP-dependent reaction catalyzed by the
C-terminal Gsp synthetase domain (supplemental Fig. S1)
(6–8). E. coli GspSA also hydrolyzes Gsp to GSH and Spd via
the N-terminal amidase domain (supplemental Fig. S1) (6).
Although GspSA was first found in E. coli more than 3 decades
ago (9), it is not known what the physiological role of Gsp is or
how the two opposing activities of GspSA are regulated in vivo.
Herein, we report that Gsp S-thiolated proteins (GspSSPs)
have mixed disulfides of Gsp and protein thiols in E. coli.
Intriguingly, we found that the level of GspSSPs increased when
E. coli was treated with H2O2, indicating that this modification
probably inhibits oxidation of protein thiols. The accumulation
of GspSSPs probably occurred because, although Gsp amidase
was inactivated by H2O2, Gsp synthetase was mostly unaf-
fected. According to an x-ray crystallography study and a
chemical modification study, we found that H2O2 oxidized the
thiol of the amidase active-site nucleophile, Cys59, to a sulfenic
acid and that the sulfenic acid was stabilized by several hydro-
gen bonds, one of which involved a water molecule and was
unusually short. We propose a set of reactions that explain how
the transient inactivation of Gsp amidase leads to an accumu-
lation of Gsp and an increased level of GspSSPs after oxidative
stress. With elimination of the oxidative threat, Gsp amidase,
GSH reductase, and glutaredoxin act in concert to convert oxi-
dized Gsp (as the disulfide of Gsp ((GspS)2), mixed disulfides of
Gsp, and other small thiol-containing compounds and/or
GspSSPs) to GSH. That GspSA and glutaredoxin act synergis-
tically is supported by the hypersensitivity of E. coli mutants
that lack both enzymes to H2O2.
Protein thiols are readily oxidized and reduced to form sulfe-
nates, sulfinates, sulfonates, and intra- and intermolecular di-
sulfides. Most organisms have complex systems that regulate the
intracellular redox states of thiols (1, 2). Small thiol-containing
biomolecules (e.g. GSH and cysteine, form mixed-disulfides
with protein thiols (S-thiolation). These post-translational
modifications protect proteins from overoxidation and regu-
late certain protein functions (3, 4). For example, S-glutathio-
nylation of Escherichia coli methionine synthase, which occurs
when E. coli is oxidatively stressed, suppresses the synthase
activity, thereby decreasing cellular methionine concentration
EXPERIMENTAL PROCEDURES
The following experiments are described in the supplemen-
tal material due to space limitations: the construction of GspSA
* This work was supported by National Science Council of Taiwan Grant
NSC97-2628-M-001-016-MY3.
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S10.
□S
1 To whom correspondence may be addressed. E-mail: whifinwu@ 4 The abbreviations used are: Spd, spermidine; (GspS)2, disulfide of gluta-
ntu.edu.tw.
thionylspermidine; GSSG, disulfide form of GSH; GSSP, GSH S-thiolated
protein; Gsp, glutathionylspermidine; GspSA, glutathionylspermidine
synthetase/amidase; Gsp-SG, Gsp glutathione mixed disulfide; GspSSPs,
Gsp S-thiolated proteins; Grx, glutaredoxin; mBBr, monobromobimane;
2-ME, ␤-mercaptoethanol; TSH, trypanothione; pNA, p-nitroanilide;
MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS,
mass spectrometry.
2 To whom correspondence may be addressed. E-mail: ahjwang@gate.
sinica.edu.tw.
3 To whom correspondence may be addressed: Institute of Biological Chem-
istry, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Tai-
wan. Tel.: 886-2-27890110; Fax: 886-2-26514705; E-mail: chunhung@
gate.sinica.edu.tw.
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Glutathionylspermidine, a Redox Regulator
disrupted strain, protein crystallization and refinement, identi- used ADP (a product of Gsp synthetase) and consumed NADH.
fication of sulfenic acid by dimedone, and the hydrolysis of NADH consumption was monitored by fluorescence emission
GspSSPs by Gsp amidase.
at 460 nm (excitation at 340 nm). The assay mixture contained
Reagents and Chemicals—The chromogenic tripeptide ␥- 125 mM Tris-HCl (pH 7.3), 0.2 mM NADH, 1 mM phosphoenol-
Glu-Ala-Gly-p-nitroanilide (␥-EAG-pNA), which was the ami- pyruvate, 10 units/ml pyruvate kinase, 10 units/ml lactate dehy-
dase substrate, was synthesized as reported (10). The thiol-la- drogenase, 2 mM ATP, 10 mM GSH, 10 mM Spd, 10 mM MgCl2,
beling reagent, monobromobimane (mBBr) and (GspS)2 were and various concentrations of H2O2. Control experiments indi-
purchased from Bachem and Invitrogen, respectively. Other cated that H2O2 did not affect NADH consumption or the
chemicals were purchased from Sigma and Merck unless spec- pyruvate kinase/lactate dehydrogenase couple.
ified otherwise.
The ability of GSH to restore amidase activity was assessed.
Protein Expression and Purification of GspSA—The genes, GspSA (0.45 nM) was first inactivated with 500 ␮M H2O2 for 5
GspSA, GspAF (residues 1–197 of full-length GspSA), GrxA min. Then catalase (final concentration 1 mg/ml) was added,
(coding for Grx1 (glutaredoxin 1)), and GrxB (coding for Grx2 and 4 mM GSH was incubated for 20 min. Inactivated Gsp ami-
(glutaredoxin 2)), were each subcloned into a pET28a plasmid dase was then incubated with the aforementioned reagents to
(Novagen). Protein expression and purification procedures fol- ascertain if they could reactivate Gsp amidase. The levels of
lowed a previous report (11).
recovered activity were measured using the ␥-EAG-pNA assay.
Detection of E. coli Gsp S-Thiolated Proteins—NR754 (wild The control experiment used H2O instead of H2O2, and its
type) and HA61002 (⌬gspSA) were each cultured in 1 ml of activity level was normalized to 100%.
M9 minimal medium at 37 °C until the A600 values of the
The Viabilities of GspSA and Glutaredoxin Null Mutants
cultures were 1.0. Then, 250 nmol of 14C-labeled spermidine after H2O2 Treatment—Overnight cultures (⌬grxA, ⌬grxB,
(GE Healthcare) was added to each culture, and they were then ⌬grxA⌬gspSA, and ⌬grxB⌬gspSA) were diluted with fresh M9
incubated for an additional 10 h. Samples of the cells were minimal medium containing 0.4% glucose and cultivated until
treated with 0.5 mM H2O2 for 30 min, collected by centrifuga- A600 of 1.0 were reached. Then different concentrations of
tion, mixed with SDS-loading buffer containing 0 or 50 mM H2O2 were added to the cultures, and they were incubated for
2-mercaptoethanol (2-ME), and then boiled for 15 min. After an additional 1 h. The cultures were serially diluted with PBS
removing cell debris by centrifugation, the samples were sub- buffer and plated onto LB agar plates. The survival percentage
jected to SDS-PAGE. The separated proteins were stained with (survivability) was calculated as ((CFU in the presence of
Coomassie Brilliant Blue or detected using a PhosphorImager H2O2)/(CFU in the absence of H2O2))/100, where CFU repre-
system BAS-1500 (Fuji, Tokyo, Japan).
sents colony-forming units. The survivability values (%) for
In Vitro and in Vivo Determination of Gsp Levels after H2O2 each H2O2 concentration were calculated using those of at least
Treatment—For the in vitro assay, a 1.8 nM GspSA solution was three replicates and are presented as the means Ϯ S.D.
first incubated with 500 ␮M H2O2 for 5 min to inactivate the
Conversion of Gsp-disulfide to GSH by a Gsp Amidase/GSH
GspSA amidase activity and then treated with catalase (final Reductase Couple—To determine if Gsp amidase could cata-
concentration 1 mg/ml) for 20 min to remove any remaining lyze the hydrolysis of (GspS)2, 5 mM Gsp-disulfide and 10 ␮M
H2O2. Omission of H2O2 served as the control. The samples GspSA (1 ␮g) were incubated at 37 °C and then subjected to
were added to 250 mM Tris-HCl (pH 7.3), 2 mM GSH, 2 mM Spd, MALDI-TOF-MS. Similarly, to determine if (GspS)2 could be
1 mM ATP, 4 mM phosphoenolpyruvate, 10 mM MgCl2, and 5 converted to GSH by the Gsp amidase/GSH reductase couple,
units/ml pyruvate kinase. Aliquots of 100 ␮l were withdrawn 0.23 unit/ml of GSH reductase (final concentration) was added
periodically and heated at 95 °C for 10 min to inactivate the to mixtures of 100 mM Tris-HCl (pH 7.3), 0.5 mM NADPH, 600
enzyme activities. Samples were then derivatized with mBBr, ␮M (GspS)2 at 25 °C with or without 28.7 ␮M GspSA. NADPH
and Gsp and GSH contents were analyzed by HPLC (12).
consumption was measured by monitoring fluorescence emis-
For the in vivo assay, E. coli BL21(DE3) was cultured in M9 sion at 460 nm (excitation at 340 nm). Replacement of (GspS)2
minimal medium until its A600 was 1.5. Then the culture was with oxidized GSH (GSSG, the native substrate of GSH reduc-
treated with 1, 5, or 10 mM H2O2. After 10 min, the cells were tase) served as the positive control, whereas the negative con-
centrifuged and lyophilized. Cells of suitable dry weight were trol did not include (GspS)2 and GSSG.
lysed, and 800 ␮M 2-ME (final concentration) was added. The
RESULTS
lysateswerederivatizedwithmBBrandthencentrifugedat8000 ϫ
g for 30 min to remove cell debris. The resulting samples were
Identification of E. coli Gsp S-Thiolated Proteins—To in-
subjected to HPLC to identify and quantify cellular thiol levels.
vestigate if Gsp S-thiolates E. coli proteins, 14C-labeled sper-
GspSA Activities Measured after H2O2 Treatment—To mea- midine (Spd*) was added to cultures of NR754 (wild type)
sure the degree to which Gsp amidase was inactivated by H2O2, and HA61002 (⌬gspSA). NR754 has an endogenous GspSA
␥-EAG-pNA was used as the substrate. The assay mixtures, at that can conjugate GSH and Spd* to form 14C-labeled Gsp
25 °C, contained 0.45 nM GspSA, 125 mM Tris-HCl (pH 7.3), (Gsp*; Fig. 1A). After uptake of Spd*, Gsp*-labeled proteins
1.25 mM ␥-EAG-pNA, and various concentrations of H2O2. were detected in lysates of NR754 by phosphorimaging after
p-Nitroanilide formation was monitored as an increase in SDS-PAGE (Fig. 1B, right). The proteins were Gsp S-thiolated
absorbance at 405 nm.
was confirmed by treatment of the Gsp*-labeled cell lysates
Gsp synthetase activity in the presence of H2O2 was assayed with 2-ME (Fig. 1B, right), which decreased the amount of
by a pyruvate kinase/lactate dehydrogenase-coupled assay that radiolabeled proteins observed and was attributed to disulfide
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Glutathionylspermidine, a Redox Regulator
cleavage by 2-ME. Proteins of
HA61002 (⌬gspSA) were not radio-
labeled because Gsp could not be
synthesized in HA61002 (Fig. 1B,
right). Interestingly, the enhanced
level of GspSSPs after H2O2 treat-
ment was reminiscent of the protec-
tive effects offered by GSH S-thiola-
tion (13), which implied that Gsp
might also protect protein thiols
against irreversible oxidation. Vari-
ous Gsp-protein conjugates were
formed (Fig. 1B, right), suggesting
that Gsp S-thiolation of proteins
may be involved in diverse types of
biological processes.
Rapid Accumulation of Gsp in
Vivo in the Presence of H2O2—To
measure the intracellular Gsp level
after treatment with H2O2, M9 min-
imal medium cultures of E. coli
BL21(DE3) that were in stationary
phase (A600 ϭ 1.2) were treated with
FIGURE 1. Detection of GspSSPs in E. coli. A, a schematic diagramming how Gsp S-thiolation of E. coli
proteins was detected. 14C-Labeled spermidine (Spd*) was added into an E. coli culture and was then
conjugated with GSH by Gsp synthetase activity in GspSA to form radiolabeled Gsp (Gsp*). Gsp*SSPs were
detected by phosphorimaging after SDS-PAGE. The levels of radiolabeled proteins were reduced when
extracts were treated with 2-ME. B, SDS-PAGE analysis of E. coli proteins in NR754 (wild type) and HA61002
(⌬gspSA). Left, Coomassie Brilliant Blue-stained proteins. Right, phosphorimaging of radiolabeled
proteins.
1, 5, or 10 mM H2O2 and then incu-
bated for an additional 10 min. The
cells were collected by centrifuga-
tion and then lysed. Monobromo-
bimane (mBBr) was added into the
lysates to derivatize thiol-contain-
ing compounds (12), and after
HPLC analysis, mBBr-modified Gsp
in the eluates was quantified (fluo-
rescence emission at 480 nm, exci-
tation at 375 nm). The addition of 1
or 10 mM H2O2 led to a 2.2- or 3.6-
fold increase in Gsp concentration,
respectively, within 10 min (Fig.
2A). Expression of GspSA was not
altered by H2O2 treatment (Fig. 2B),
indicating that the observed
increase in Gsp was not linked to the
level of GspSA.
Selective Inactivation of Gsp Ami-
dase by H2O2—To determine how
the GspSA in E. coli is affected by
oxidative damage, the enzyme was
treated with 50, 100, and 500 ␮M
H2O2, and the synthetase and ami-
dase activities were determined. In
all cases, the Gsp amidase activity
decreased as functions of time
and dose (Fig. 3A) with 50% inhib-
itory concentration of 250 ␮M (data
not shown). Inactivation was Ͼ95%
when GspSA was incubated with
500 ␮M H2O2 for 5 min. Conversely,
the synthetase activity remained
almost completely active under the
FIGURE 2. Gsp accumulation in E. coli after H2O2 treatment. The levels of Gsp and GspSA in E. coli were
measured after the bacteria had been cultured in M9 minimal medium, treated with H2O2, lysed, and treated
with mBBr. A, HPLC chromatograms of mBBr-derivatized thiol compounds detected using fluorescence spec-
troscopy. The arrows indicate the elution position of Gsp. The numbers above the arrows are the amounts
(␮mol) of Gsp obtained from 1 mg, dry weight, of cells. B, SDS-PAGE of GspSA shows that its expression level
was unaffected by H2O2.
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Glutathionylspermidine, a Redox Regulator
FIGURE 3. Effects of H2O2 on GspSA amidase and synthetase activities. A, plots of Gsp amidase activity that remained after treatment with four different
concentrations of H2O2. Activity was measured using the chromogenic substrate ␥-EAG-pNA. B, plots of Gsp synthetase activity that remained after treatment
with four different concentrations of H2O2. At worst, there was only a 10% reduction in synthetase activity compared with that of the negative control (0 M
H2O2). Synthetase activity was measured as the consumption of NADH by the pyruvate kinase/lactate dehydrogenase couple, which also used ADP generated
by Gsp synthetase.
same conditions (Fig. 3B). The finding that H2O2 affects the two (H2Otriad; Fig. 4B and supplemental Table S1); therefore, the
activities differently is supported by the structural information extra electron density probably arose as a consequence of an
described below.
S-O1ꢀꢀꢀHꢀꢀꢀO2-H hydrogen bond. Partial negative charges could
To determine whether H2O2-mediated amidase inactivation be present at O1 and O2 because such hydrogen bonds have
could be rescued by a biological reducing agent, inactivated partial ionic character. Conversely, we do not believe that a
GspSA (treated with 500 ␮M H2O2 for 5 min and then incubated hydroperoxide-derivatized cysteine (–S-O–O-H) was present
with catalase for 20 min to remove excess H2O2) was exposed to because the S-O and O-O distances are not compatible with such
GSH. The addition of 4 mM GSH (physiological concentration) a compound, and it would be less stable than a sulfenic acid (15).
recovered ϳ80% of the amidase activity (supplemental Fig. S2).
The sulfenic acid that was produced when a GspAF crys-
To examine how selectively inactivated Gsp amidase affected tal was soaked with 1 mM H2O2 for 20 min was reduced to a
the level of Gsp, GspSA was treated with GSH, Spd, ATP, phos- free thiol when subsequently treated with 1 mM GSH (data
phoenolpyruvate, and pyruvate kinase with or without prior not shown). Interestingly, the sulfenic acid was not further
exposure to H2O2. The mixtures were then treated with mBBr oxidized by either prolonged incubation with H2O2 or by a
and analyzed by HPLC. Whether H2O2-treated or not, the Gsp greater concentration of H2O2 (10 mM). To our surprise, Cys59
concentrations initially increased and then declined (supple- was covalently bonded to an acetate oxygen in the GspAF_ac-
mental Fig. S3); therefore, the level of Gsp was tightly regulated. etate crystal structure (Protein Data Bank code 3A30; supple-
After pretreatment with H2O2, Gsp accumulated to a greater mental Fig. S4). This S-modification was probably caused by a
extent than it did otherwise (no treatment). Additionally, less nucleophilic attack of an acetate ion on the sulfenic acid, which
GSH was present at all times longer than the initial stage (ϳ25 was favorable because of the large acetate concentration (100
min). Both of these observations suggested that H2O2 selec- mM) and the prolonged exposure.
tively inactivates Gsp amidase, but the amidase activity recov-
ers at a later stage. More Gsp was produced as a consequence of amidase, was oxidized by H2O2, other GspSA cysteines, includ-
Although Cys59, which is the catalytic nucleophile for GspSA
oxidative damage.
ing Cys338 and Cys539, which form part of the synthetase bind-
Identification of Cys59-Sulfenic Acid by X-ray Crystallogra- ing site (9), were not oxidized when crystals of GspSA were
phy and a Chemical Modification Study—The structure of the treated with H2O2 (data not shown).
H2O2-treated Gsp amidase fragment (GspAF_H2O2; supple-
To further corroborate that the Cys59 thiol was oxidized to a
mental Table S1) was obtained by soaking a GspAF crystal in sulfenic acid (16), GspAF was first treated with 100 ␮M H2O2 for
crystallization buffer containing 1 mM H2O2 for 3 days before 10 min, which fully inactivated GspAF, and then reacted with
x-ray crystallography. The electron density map of GspAF_ 100 ␮M 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which
H2O2 (Protein Data Bank entry 3A2Z) at 1.5 Å resolution has a specifically reacts with sulfenic acid (16), at 25 °C for 12 h. After
density not seen in untreated GspAF that extends from Cys59
S
removing excess reagents, derivatized GspAF was digested with
␥
and corresponds to two adjacent oxygen atoms (Fig. 4A). The trypsin, and its hydrolytic products were analyzed by MALDI-
ϩ
distance between the S and O1 is 2.1 Å, the C-S–O1 angle is TOF-MS (Fig. 4C). The signal (M ϩ H) at m/z 1176.8
96.0°, and the S-O1-O2 angle is 94.2°. The second oxygen atom is expected for the adduct formed by the GspSA sequence
(O2) is unusually close to O1 (2.2 Å). Using the structural and 57WQCVEFAR64 (Mr ϭ 1037) and dimedone (Mr ϭ 140). The
other information (see below), we suggest that the Cys59 thiol presence of b- and y-series ions confirmed that dimedone mod-
was oxidized to sulfenic acid and that its oxygen atom forms a ified the Cys59 sulfenic acid. Therefore, the thiol of Cys59 was
very short hydrogen bond (Ͻ2.3 Å) (14) with a water molecule oxidized to the sulfenic acid by H2O2.
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Glutathionylspermidine, a Redox Regulator
FIGURE 4. Identification of the Cys59 sulfenic acid by x-ray crystallography and chemical modification/mass spectrometry. A, stereo view of the
2Fo Ϫ Fc electron density map at the active site of GspAF_H2O2. The electron density map, drawn at a contour level of 1␴, shows continuous density (in
red) connected to the S␥ atom of Cys59. This density was fitted with the oxygen atom of a sulfenic acid (-SOH) and the oxygen of a tightly hydrogen-
bonded water molecule. Limited by the structure resolution, it is possible that the Asn149 side chain can be flipped. B, schematic of the hydrogen bond
network associated with the Cys59 sulfenic acid in GspAF_H2O2. The sulfenic acid/water hydrogen bond is shown in red. Two other water molecules that
contribute to the stability of the sulfenic acid are shown in blue. C, MALDI-MS-MS spectrum of the tryptic peptides that contained the Cys59-dimedone
adduct. Gsp amidase, after oxidation by H2O2, was treated with dimedone, digested with trypsin, and subjected to MALDI-MS. The dimedone-modified
Cys59-containing peptide (57WQCVEFAR64) has a molecular weight of 1176.6. That covalent dimedone modification of Cys59 had occurred is evidenced
by the y- and b-ions identified with asterisks.
Hydrolysis of Gsp-disulfide and Gsp S-Thiolated Proteins formed to determine if (GspS)2 was a substrate for GSH reduc-
and Peptides by Gsp Amidase—E. coli lacks an enzyme that tase. GSH reductase activity was detected as NADPH con-
can reduce (GspS)2 (17). If (GspS)2 is involved in E. coli sumption, which was monitored by NADPH absorption at 340
redox regulation, then there must be a way to convert nm (Fig. 5B). GSH reductase alone (without the addition of
(GspS)2 to Gsp. We next showed that it is possible to convert GSSG) served as the negative control (open circles in Fig. 5B).
(GspS)2 to GSH by coupling Gsp amidase and GSH reductase No activity was observed when (GspS)2 was incubated with only
activities. (GspS)2 could be hydrolyzed by Gsp amidase, as GSH reductase and NADPH (filled squares), in contrast to the
shown by MALDI-TOF-MS analysis. After a 10-min or 4-h result found for GSSG (filled circles). Activity was also observed
incubation (GspS)2 with GspSA, Spd, Gsp-GSH mixed disul- when (GspS)2 was treated with GspSA and GSH reductase
fide (Gsp-SG), and GSH disulfide (GSSG) were generated, as (open triangles). The experimental results presented in Fig. 5B
indicated by the presence of MS peaks at m/z 740.6 ((M ϩ suggested that both (GspS)2 and Gsp-SG were converted to
ϩ
ϩ
H) , Gsp-SG), at m/z 613.3 ((M ϩ H) , GSSG), and at m/z GSH and Spd via consecutive reactions by Gsp amidase and
ϩ
635.3, ((M ϩ Na) , GSSG) (Fig. 5A). A second assay was per- GSH reductase (Fig. 5C).
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Glutathionylspermidine, a Redox Regulator
FIGURE 5. Conversion of (GspS)2 to GSH and Spd by the Gsp amidase/GSH reductase couple. A, a MALDI-TOF spectrum that shows the molecular weights
of the hydrolysis products after (GspS)2 was treated with GspSA for 0 min (top), 10 min (middle), or 4 h (bottom). GSSG was the end product. B, the Gsp
amidase/GSH reductase coupled assay. (GspS)2 cannot be reduced by GSH reductase alone (f), but it is reduced when both GspSA and GSH reductase are
present (‚). GSSG plus GSH reductase served as the positive control (F). GSH reductase alone served as the negative control (E). C, reaction scheme for the
enzymatic conversion of (GspS)2 to GSH and Spd by the Gsp amidase/GSH reductase couple.
We next tested whether Gsp S-thiolated peptides and pro- reached mid-exponential phases, at which point they were
teins were substrates for Gsp amidase. Lysates of E. coli treated with H2O2, incubated for 1 h, serially diluted with
proteins were treated with disulfide of biotinated Gsp ((Bio- PBS buffer, and then transferred to LB plates. The viabilities
tin-GspS)2) (Fig. 6A) to generate biotin-labeled GspSSPs of NR754 and HA61002 under oxidative stress were similar
(biotin-GspSSPs), which could be detected with an anti- (supplemental Fig. S6), which is consistent with a previous
biotin antibody (Fig. 6B). Treatment of these proteins with report that an E. coli strain lacking the ability to synthesize
GspSA removed Spd-biotin moieties because biotin-GspSSPs GSH was as resistant to H2O2 as the corresponding wild-type
were no longer observed after staining with the anti-biotin strain was (18).
antibody (Fig. 6C, left panel, lane 3). Treatment with 2-ME
Because glutaredoxins can reduce GSH S-thiolated pro-
also produced the same result, presumably because 2-ME teins (GSSPs) (19), we wanted to determine if these enzymes are
cleaved Gsp-protein disulfides (Fig. 6C, left panel, lane 2). A part of a redox cycle involving Gsp S-thiolation. The viabilities
synthetic Gsp-containing peptide (T28-Gsp) was also incu- of two glutaredoxin null strains (⌬grxA and ⌬grxB) and two
bated with GspSA, and a MALDI-TOF-MS spectrum of that double mutant strains (⌬grxA⌬gspSA and ⌬grxB⌬gspSA) were
incubation mixture indicated that T28-Gsp was a substrate examined after they were treated with H2O2. When preincu-
for Gsp amidase (supplemental Fig. S5). Therefore, Gsp ami- bated with 1 or 5 mM H2O2 for 1 h, ⌬grxA and ⌬grxB had
dase can hydrolyze a variety of Gsp-derivatized substrates, survivability values of 60 and 30%, respectively (Fig. 7). The
yielding Spd and GSH S-thiolated proteins/peptides.
double mutant strains were even more susceptible to oxidative
Sensitivities of Different E. coli Strains to Oxidative Stress— damage because their survivabilities were reduced by 70 and
To characterize how Gsp and GspSA protect against oxidative 99% after exposure to 1 and 5 mM H2O2, respectively. There-
stress, cultures of NR754 (wild type) and HA61002 (⌬gspSA) fore, there is a synergistic protective effect by GspSA and Grx
were treated with various concentrations of H2O2, and the against oxidative damage. Moreover, in a separate experiment,
viabilities of the cells were examined after treatment. The the levels of GspSSPs were substantially decreased when
strains were cultured in M9 minimal medium until the cells recombinant glutaredoxin (Grx1 or Grx2) was added to E. coli
25350 JOURNAL OF BIOLOGICAL CHEMISTRY
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Glutathionylspermidine, a Redox Regulator
consequently causes Gsp to accu-
mulate (Fig. 8). Accumulated Gsp
possibly scavenges oxidants directly
or forms mixed disulfides with pro-
tein thiols. Once the source of the
oxidative stress has been elimi-
nated, Gsp amidase activity can be
rescued by reaction of the sulfenic
acid with a reducing reagent (e.g.
GSH or Gsp). Sulfenic acid is a reac-
tive electrophile and reacts with
thiol reagents, such as GSH, to gen-
erate mixed disulfides. The amidase
active site is solvent-exposed, which
should allow GSH or other small
thiol-containing molecules to react
with the sulfenic acid. After forma-
tion of a mixed disulfide, an addi-
tional thiol exchange may continue
regenerating the thiol of Cys59 and
thereby recovering amidase activity
(supplemental Fig. S8). The reacti-
vated amidase may then hydrolyze
either excessive Gsp to GSH and
Spd and/or GspSSPs to GSSPs and
Spd. Gsp could also be oxidized to
produce (GspS)2 and/or other mixed
disulfides upon reaction with reactive
oxygens. We showed that GSH re-
ductase alone cannot reduce (GspS)2
but that Gsp amidase must first re-
move the spermidine moiety. The
FIGURE 6. Conversion of GspSSPs to GSSPs by Gsp amidase-catalyzed hydrolysis. A, the (biotin-Gsp)2 conversion of (GspS)2 to Spd and
structure. B, schematic showing the preparation of biotinylated Gsp S-thiolated proteins (biotin-GspSSPs).
Treatment of biotin-GspSSPs with GspSA generated GSH S-thiolated proteins. Treatment of biotin-GspSSPs
with 2-ME generated unlabeled proteins. C, SDS-PAGE of biotin-GspSSPs. Right, Coomassie Brilliant Blue-
stained proteins. Left, Western blot using an alkaline phosphatase-conjugated anti-biotin antibody. Mr, molec-
ular weight markers. Lane 1, biotin-GspSSPs from an E. coli lysate. Lane 2, biotin-GspSSPs as in lane 1 that were
treated with 20 mM 2-ME before SDS-PAGE. Lane 3, biotin-GspSSPs as in lane 1 that were treated with 5 ␮g of
GspSA before SDS-PAGE.
GSH by the Gsp amidase/GSH reduc-
tase couple suggests that Gsp may be
part of an oxidative defense mecha-
nism that does not require a Gsp-
specific reductase. Such a reductase
has not been found in E. coli (17).
The results allow us to propose a reaction scheme (Fig. 8) that
explains how Gsp amidase activity is modulated in vivo so that
the Gsp level reflects the intracellular redox condition.
lysates that contained Gsp*SSPs (supplemental Fig. S7). These
results strengthen the idea that Grxs participate in the reduc-
tion of Gsp S-thiolated proteins.
Glutaredoxins reduce mixed disulfide bonds between
GSH and GSSPs by forming a GrxSSG intermediate that can
DISCUSSION
Redox Regulation of Gsp and Gsp S-Thiolated Proteins— be subsequently reduced by a molecule of GSH (20). The
Most organisms use GSH to regulate their intracellular thiol protein expression level of Grx1 is enhanced under oxidative
and disulfide levels. Gsp is a GSH derivative found only in some stress in E. coli, whereas Grx2 is constitutively produced
bacteria and parasitic protozoa. As reported herein, we showed (21). We found that the amounts of GspSSPs decreased in the
that Gsp forms mixed disulfides with the thiols of E. coli pro- presence of Grx1 or Grx2 (supplemental Fig. S7), suggesting
teins in vivo and that the amounts of these Gsp derivatives are that Grxs can reduce mixed disulfides. As mentioned, Gsp ami-
linked to intracellular redox conditions. In vivo Gsp S-thiola- dase catalyzes the hydrolytic removal of Spd from GspSSPs,
tion of E. coli proteins is affected by several factors, including leading to the formation of GSSPs. A Grx can then reduce the
the intracellular Gsp concentration. We demonstrated that the GSSPs to generate free protein thiol(s) (supplemental Fig. S9).
Gsp level increases when E. coli is oxidatively stressed. The However, we cannot exclude the possibility that glutare-
selective inactivation of Gsp amidase provides a rational expla- doxin directly reduces GspSSPs, with the resulting Grx-Gsp
nation for this event. When E. coli is exposed to reactive oxygen mixed disulfide intermediate subsequently reduced via thiol-
species, the thiol of Cys59 of the active-site amidase domain is disulfide exchange (supplemental Fig. S9). Because ⌬grxA⌬gspSA
oxidized to sulfenic acid, which inactivates Gsp amidase and and ⌬grxB⌬gspSA were more easily killed by H2O2 than were
AUGUST 13, 2010•VOLUME 285•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 25351

Glutathionylspermidine, a Redox Regulator
⌬grxA and ⌬grxB, it appears that the activities of Grx and
To date, most studies concerning protein S-thiolation
GspSA can provide a coordinated defense against oxidative have focused on GSH, because GSH is abundant in cells
damage in E. coli.
(4). However, protein S-thiolation by other thiol-containing
molecules, such as cysteine or Gsp, may have different ef-
fects. For example, a previous study demonstrated that GSH
S-thiolation of Ca2ϩ-dependent protein kinase C␣ inhibits
the activity of Ca2ϩ-dependent protein kinase C␣ and its
isozymes, whereas cysteine S-thiolation does not (22). Both
trypanothione (TSH) and Gsp are more efficient reducing
agents than is GSH. The non-enzymatic reductions of dehy-
droascorbate and H2O2 by Gsp and TSH are several times
faster than those by GSH (23, 24). It has also been found that
Gsp was a more efficient S-thiolating agent than was GSH
(25). GSH is negatively charged (Ϫ1) at physiological pH,
whereas Gsp is positively charged (ϩ2). Gsp and GSH there-
fore introduce opposite charges into the proteins that they
thiolate. Protein thiols are deprotonated to form thiolates
that interact more readily with (GspS)2 at pH Ͼ6, rather than
GSSG (25). Although GSH and Gsp have distinct physical
and chemical properties, it is still difficult to elucidate how
GSH and Gsp function in vivo because of the involvement of
the GspSA amidase-catalyzed conversion of GspSSPs to
GSSPs.
FIGURE 7. Glutaredoxin- and GspSA/glutaredoxin-null mutants exhibit
hypersensitivities to exogenous H2O2. All strains were grown in M9 mini-
mal medium until the cell densities reached 1.0 A600. Then the cultures were
treated with various concentrations of H2O2 for 1 h, and then 0.1 ml of each
culture was transferred onto LB plates. The numbers of viable cells were then
determined. Survival percentages (survivabilities) of ⌬grxA (‚, blue), ⌬grxB
(Œ, black), ⌬grxA⌬gspSA (E, brown), and ⌬grxB⌬gspSA (F, red) were calcu-
lated as ((CFU in the presence of H2O2)/(CFU in the absence of H2O2))/100.
Survivability values (%) are the means of three replicates Ϯ S.D. (error bars).
Dissimilar Amidase Active Sites Linked to Differential Redox
Regulation—Parasitic protozoa use TSH, which is synthesized
by TSH synthetase/amidase to defend against oxidative dam-
age. Like GspSA, TSH synthetase/amidase has an amidase
activity that can hydrolyze the amide bond of Gsp. There-
fore, it would be interesting to
know if TSH amidase can be selec-
tively and reversibly inactivated.
In the x-ray structure of Leishma-
nia major TSH synthetase/amidase,
the C-terminal segment partially
obstructs accessibility to the cata-
lytic Cys59 nucleophile (26). Con-
sequently, the active site of TSH
amidase is substantially blocked, in
contrast with that of GspSA, which
is part of a large, solvent-accessible
cleft. Even if the TSH amidase
Cys59 thiol could be oxidized to
sulfenic acid and/or form a mixed
disulfide with GSH or TSH, the
restricted access almost certainly
would impede subsequent thiol
exchange (supplemental Figs. S8
and S10). Because access to the TSH
amidase active site is partially
obscured, TSH reductase and TSH
tryparedoxin may compensate as a
defense mechanism against reactive
FIGURE 8. The roles of Gsp and glutathionylspermidine synthetase/amidase in the intracellular redox
regulation of E. coli. When exposed to reactive oxygen species (ROS), the active-site Cys59 thiol of Gsp ami-
dase is oxidized to sulfenic acid (1), which causes the inactivation of Gsp amidase (ϫ) (2). Because Gsp synthe-
tase is not affected by ROS, intracellular Gsp accumulates (3). Gsp may then scavenge harmful oxidants by
forming Gsp-disulfides and other small molecule disulfide compounds (4A) and/or protecting protein thiols
fromoxidationbyGspS-thiolation(4B). Withtheremovaloftheoxidativestresses, intracellularGSHand/orGsp
mayrescueoxidizedGspamidase, restoringamidaseactivity(4C). ReactivatedGspamidasemayhydrolyzeGsp
to GSH and Spd or hydrolytically remove Spd from Gsp-disulfide or Gsp-modified proteins. Finally, the amount
of Gsp returns to its basal level (5).
oxygen in parasitic protozoa. It is
also possible that, with the evolu-
tionary emergence of TSH reduc-
tase and TSH tryparedoxin, TSH
amidase degenerates and no longer
participates in redox regulation.
25352 JOURNAL OF BIOLOGICAL CHEMISTRY
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Glutathionylspermidine, a Redox Regulator
Cys59 Sulfenic Acid of GspSA Is Primarily Stabilized by a
Very Short Hydrogen Bond—Sulfenic acid is an unstable
compound and is highly reactive; therefore, it usually acts as
an intermediate en route to other more stable oxidized sulfur
compounds (e.g. sulfinic and sulfonic acids) (27). Nature has
used different strategies to stabilize sulfenic acids. For exam-
ple, the sulfur of sulfenic acid and an amide nitrogen can
form a cyclic sulfonamide, which has been found in the crys-
tal structure of protein-tyrosine phosphatase (Protein Data
Bank entry 1OEM) (28). The sulfenic acid of an archaeal
2-Cys peroxiredoxin (Protein Data Bank entry 2ZCT) is
Acknowledgments—Some E. coli strains were obtained from the
National Bioresource Project, Japan. We thank the National Synchro-
tron Radiation Research Center of Taiwan and the National Core
Facility of Proteomics for mass spectrometry analysis.
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the amidase activity was restored. Moreover, the hypersensitiv-
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AUGUST 13, 2010•VOLUME 285•NUMBER 33
JOURNAL OF BIOLOGICAL CHEMISTRY 25353

Enzymology:
Protein S-Thiolation by
Glutathionylspermidine (Gsp): THE ROLE
OF ESCHERICHIA COLI Gsp
SYNTHETASE/AMIDASE IN REDOX
REGULATION
Bing-Yu Chiang, Tzu-Chieh Chen, Chien-Hua
Pai, Chi-Chi Chou, Hsuan-He Chen, Tzu-Ping
Ko, Wen-Hung Hsu, Chun-Yang Chang,
Whei-Fen Wu, Andrew H.-J. Wang and
Chun-Hung Lin
J. Biol. Chem. 2010, 285:25345-25353.
doi: 10.1074/jbc.M110.133363 originally published online June 8, 2010
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