New Thioredoxins and Glutaredoxins as Electron Donors of
3'-Phosphoadenylylsulfate Reductase*
Christopher Horst
Lillig
,
Antje
Prior
,
Jens Dirk
Schwenn
§,
Fredrik
Åslund¶,
Daniel
Ritz¶,
Alexios
Vlamis-Gardikas
, and
Arne
Holmgren
From the
Biochemistry of Plants, Faculty of Biology,
Ruhr-University, 44780 Bochum, Germany, ¶ Department of
Microbiology and Molecular Genetics, Harvard Medical School, Boston,
Massachusetts, and
Medical Nobel Institute for Biochemistry,
Department of Medical Biochemistry and Biophysics, Karolinska
Institute, Stockholm, Sweden
 |
ABSTRACT |
Reduction of inorganic sulfate to sulfite in
prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as
substrate for PAPS reductase and is the first step leading to reduced
sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly
identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2,
Grx3, and a mutant Grx1C14S) was compared with the well known
thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a
Vmax ranging from 6.6 units mg
1
(Trx1) to 5.1 units mg
1 (Grx1), whereas Grx1C14S was only
marginally active, and Grx2 and Grx3 had no activity. The structural
difference between active reductants had no effect upon
Km PAPS (22.5 µM). Grx1 effectively replaced Trx1 with essentially identical
Km-values: Km trx1
(13.7 µM), Km grx1 (14.9 µM), whereas the Km trx2
was considerably higher (34.2 µM). The results agree with
previous in vivo data suggesting that Trx1 or Grx1 is
essential for sulfate reduction but not for ribonucleotide reduction in
E. coli.
 |
INTRODUCTION |
Prototrophic bacteria or fungi mainly use inorganic sulfate as the
only supply of sulfur for the biosynthesis of amino acids and essential
cofactors. Assimilation of sulfate occurs in five enzymatic steps.
First, it is activated to adenylylsulfate
(APS)1 and
3'-phosphoadenylylsulfate (PAPS) by ATP sulfurylase and APS kinase.
PAPS is then reduced to sulfite by PAPS reductase and sulfite is
reduced to sulfide by sulfite reductase. Finally, cysteine is formed
when sulfide is incorporated into
O-acetyl-L-serine (OAS) by OAS-(thiol)lyase. The
enzyme 3'-phosphoadenylylsulfate (PAPS) reductase (EC 1.8.99.4)
catalyzes the first reductive step in this sequence. It uses
thioredoxin (Trx) or glutaredoxin (Grx) as in vitro hydrogen
donor for the reduction of 3'-phosphoadenylylsulfate to free
sulfite.
|
(Eq. 1)
|
|
(Eq. 2)
|
The requirement for a low weight dithiol in sulfite formation from
PAPS was originally described by Wilson et al. (1). Gonzalez-Porqué et al. (2) identified thioredoxin as
reductant while investigating methionine sulfoxide and sulfate
reduction in yeast. Glutaredoxin as the alternate cofactor was found by Tsang and Schiff (3) in a thioredoxin-negative mutant. In
Escherichia coli, thioredoxin or glutaredoxin is essential
for sulfate reduction but not for the reduction of ribonucleotides as
deletion or inactivation of both the genes trxA and
grxA caused cysteine auxotrophy but allowed growth on
minimal medium (4). When supplied with cysteine the Trx-Grx double
mutants were still viable implying that they contained a reductant that
could substitute for thioredoxin or glutaredoxin in ribonucleotide
reduction. The search for alternate reductant(s) led the isolation of
two new glutaredoxins, termed Grx2 and Grx3 (5) and later a hitherto
unrecognized larger heat labile thioredoxin, Trx2 (6). The yet to be
explored function of the new redoxins as alternate reductants of
ribonucleotide reductases (7), their role in the maintenance of
compartmental homeostasis (8), and role in the protection against
oxidative stress (9) re-addresses also the question to their possible function as electron donors of the PAPS reductase. In addition to the
original observations by Gonzalez-Porqué et al. (2) and Tsang (10), that thioredoxin and glutaredoxin can serve as
reductant in partially purified preparations, cumulative evidence from
earlier investigations suggested that PAPS reductase from E. coli could in fact be used to detect thioredoxins from different origins (11) and of different biochemical properties (12).
More recently a homogeneous PAPS reductase from E. coli was
investigated in greater detail defining its reaction mechanism (13) and
its three-dimensional structure (14). Structural and functional data
established a ping-pong mechanism for the enzyme homodimer in which
thioredoxin reduced the enzyme in a first reaction forming a stable
reduced enzyme isoform that is oxidized by PAPS as a second reactant to
give sulfite and adenosine 3',5'-bis-phosphate (PAP). A highly
conserved cysteine residue in an ECGLH-motif near the C terminus was
recognized as the reactive nucleophile of the enzyme. No evidence for
an intermediary thioredoxin S:SO3 or a catalytically
competent enzyme substrate complex was obtained making thioredoxin
operate as a protein disulfide reductase. PAPS reductase is specific
for PAPS and thioredoxin as the electron donor cannot be replaced by
monothiols like glutathione or artificial dithiols like dithiothreitol.
Until today, all previous data concerning thioredoxin as the electron
donor of the PAPS reductase were obtained with a protein that is now
designated as thioredoxin 1 (Trx1). In this study, we could extend
these investigations using glutaredoxin1 (Grx1) and the newly
discovered glutaredoxin 2 (Grx2), glutaredoxin 3 (Grx3), and
thioredoxin 2 (Trx2). The major aim was to compare their function as
substrate of PAPS reductase and to evaluate their specificity as
hydrogen donor.
 |
EXPERIMENTAL PROCEDURES |
Bacteria Strains, Plasmids, and Oligonucleotides--
E.
coli TG1: supE hsd
5 thi
(lac-proAB) F'(traD36
proAB+ lacIq
lacZ
M15); BL21(DE3): F', ompT,
hsdSB
(rB
rM
), gal,
dcm (DE3) (Novagen, Heidelberg). Plasmid
pET16bcysH is a derivative of pET16b (Novagen) used for
cloning of a 733-base pair cysH polymerase chain reaction
fragment from pUB5 (13) amplified by polymerase chain reaction using
the oligonucleotides PRNdeI (5'-GTGAGGAACATATGTCCAAAC-3')
and PRBglIIrev (5'-CCGGCAAGA-TCTACCCTTCG-3').
Growth of Bacteria, Expression, and Purification of PAPS
Reductase--
Transformed E. coli BL21(DE3) cells were
grown in complete LB medium at pH 7.5 containing 100 µg · ml
1 ampicillin (15). The medium was supplemented with 2%
glycerol when the bacteria were grown in a fermenter. The fermenter
(Meredos, Göttingen) was inoculated with a mid log phase culture
of E. coli BL21(DE3) at a concentration of 2% (v/v)
harboring plasmid pET16bcysH. Cells were grown under
vigorous aeration at 32 °C. Expression of cysH was
induced by adding isopropyl-
-D-thiogalactoside to a
final concentration of 0.5 mM when the cells had reached a
density of A595 0.7. The cells were collected
within 2-3 h after induction by centrifugation at 10,000 × g for 20 min and washed in 20 mM Tris/HCl, 100 mM NaCl, pH 8.0. The yield was 2.5 g
liter
1. Bacteria (0.06 g ml
1 in 20 mM Tris/HCl, 100 mM NaCl, pH 8.0) were
disrupted by two passages through a French press (Ribi Cell
Fractionator) applying 14.2 MPa at 5 °C. The homogenate was
clarified by centrifugation at 15,000 × g for 20 min
and applied to TALON affinity resin (CLONTECH, Heidelberg). His10-tagged protein was eluted with 200 mM imidazole. Its purity was examined by SDS-PAGE according
to Laemmli (16) and laser mass spectroscopy (17). The average yield per
liter of broth was 14 mg PAPS reductase using LB medium. Recombinant Trx1 was purchased from MBI Fermentas (St.Leon-Roth). Trx2 was expressed and purified using the method of Miranda-Vizuete et al. (6). Grx1, Grx1C14S, Grx2, and Grx3 were purified as described earlier (18-21).
PAPS Reductase--
PAPS reductase activity was measured as
formation of [35S]-SO32
from
[35S]PAPS (12). Each individual determination of the
reaction velocity was averaged from two identical samples. The kinetic
constants were calculated from three sets of data with the lowest error derived from six different concentrations of PAPS versus
five different concentrations of reductant. The assay mixture contained 100-750 ng ml
1 of purified PAPS reductase, 100 mM Tris/HCl, pH 8.0, 10 mM
Na2SO3, 0.5-60 µM
[35S]PAPS (specific radioactivity: 1700 Bq × nmol
1), 0.5-50 µM thioredoxin kept reduced
by 10-25 mM dithiotreitiol, or 0.5-50 µM
glutaredoxin kept reduced by 10-25 mM reduced glutathione. [35S]PAPS was prepared enzymatically from
[35S]SO42- (Amersham-Buchler,
Braunschweig) using recombinant His10-APS kinase from
Arabidopsis thaliana (22). The reaction system contained ATP-sulfurylase and inorganic pyrophosphatase from Sigma
(München), pyruvate kinase and phosphoenolpyruvate from
Boehringer Mannheim as described by Schriek and Schwenn (23). PAPS
synthesis was monitored by high performance liquid chromatography as
described by Schwenn and Jender (24).
General Methods--
Isolation, restriction, and cloning of DNA,
agarose gel electrophoresis, and polymerase chain reaction were
performed according to Sambrook et al. (15). E. coli BL21(DE3) cells were transformed according to Hanahan (25).
DNA sequencing was done with an automated DNA sequencer using Auto Read
sequencing kit (Pharmacia, Freiburg) with fluorescein-labeled primers.
Annealing temperatures for oligonucleotides were calculated depending
on their G:C content. The concentration of proteins was determined
colorimetrically using Coomassie Brillant Blue (26) or from a Warburg
and Christian nomogram (27). SDS-PAGE was run on 12.5% gels replacing
-mercaptoethanol by dithiothreitol. Polyclonal anti-PAPS reductase
antibodies from rabbit (28) were used to detect the protein by Western
immunoblotting (29). Immunoprecipates were visualized on nitrocellulose
BA-S 85 (Schleicher & Schuell) by peroxidase-conjugated goat
anti-rabbit antibodies. 4-Chloro-1-naphtol in methanol was used for staining.
 |
RESULTS AND DISCUSSION |
PAPS reductase as a His10-tag fusion protein was
purified by chelate affinity chromatography yielding a homogeneous and
enzymatically active protein in a single step. The method used
previously by Berendt et al. (13) involved several
chromatographic steps including hydrophobic interaction chromatography
on phenyl-Sepharose and dye matrix affinity chromatography on blue- and
red-Sepharose. The His-tagged fusion protein is pure as judged by
SDS-gel electrophoresis and Western immunoblotting using polyclonal
antibodies against the recombinant protein (Fig.
1, A and B). By
laser mass spectroscopy (17), we could confirm that the
His10-tagged PAPS reductase was expressed as unmodified
protein with a mono-isotopic mass of 30634.4 as expected (data not
shown). The mass spectroscopy also indicated the presence of a
by-product with a mass peak that differed by 149.3 daltons. It is
assumed that the difference in isotopic mass of this by-product very
likely was caused by a His10-tag PAPS reductase lacking the
N-terminal methionine.

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Fig. 1.
Recombinant expression of PAPS
reductase. A, SDS-PAGE (12.5% Coomassie Blue) of cell
extracts from E. coli BL21(DE3). B, Western blotting
of the SDS gel using anti-PAPS reductase antibodies. Lane 1,
host organism without plasmid; lane 2, pET16b; lane
3, pET16b with cysH inserted; lane 4, 3 µg
of authentic PAPS reductase (PR).
|
|
Optimized reaction conditions for the PAPS reductase were found at a pH
value of 9.2-9.3. The rate of reaction increased by a factor of 1.93 per
10 °C interval from 18 to 35 °C provided that the
concentration of enzyme was maintained below 750 ng ml
1
of purified protein. Under these conditions, less than 20% of the
substrate is converted if the reaction time is kept
180 s (data not
shown). Rates obtained under these conditions were taken as initial
velocity. The molecular activity of the His10-tagged protein was 3.5 s
1 per catalytic center compared with
0.05 s
1 reported earlier for the recombinant untagged
enzyme (13). We know now that the lower specific activity of the
recombinant untagged enzyme was due to a high proportion of inactive
protein, which copurified with the intact enzyme protein. Laser mass
spectroscopy and refinement of the three-dimensional structure showed
that this truncated protein lacked fourteen amino acids of the C
terminus including the functionally important cysteine, whereas
thirteen residues at the truncated terminus were disordered and could
not be located (14). We have reason to believe that this truncated protein was already produced by the host TG1 transformed with the pBTac
derivative pUB5. The new His10-tagged protein described here was produced in BL21(DE3) using a pET16b vector (Novagen) giving
rise to a complete and enzymatically active PAPS reductase without
removal of the His10-tag fusion.
The Michaelis constants were determined with the concentration of PAPS
near saturating (2.5-30 µM) and that of Trx1 slightly limiting to near saturating (1-20 µM). Double reciprocal
plots produced a set of parallel lines indicating a ping-pong type
reaction mechanism. The set of lines produced a common intersect on the y axis above the origin when the data were plotted in a
Hanes-Woolf plot. The slopes were replotted to give
Vmax and Km (Fig. 2, A and B).
Kinetic constants for Grx1 were determined accordingly (Fig.
3, A and B).
In vitro, Trx1 is effectively replaced by Grx1, which
supported the rate of sulfite formation at a comparable rate (6.8-6.6
units mg
1 versus 5.4-4.8 units
mg
1 using Grx1). A lack of effect upon the Michaelis
constant for PAPS may have been expected, because only the reduced
enzyme isomer can react with the substrate but it is also noteworthy
that the Km values for Trx1 or Grx1 were virtually
identical. When thioredoxin and glutaredoxin are compared as reductants
of the ribonucleotide reductase, Grx1 with its 10-fold lower
Km, appeared to be more predominant in
vivo. It also appears more specific because it did not reduce
structural disulfides though both reductants were reported to support a
similar Vmax (30). Yet, the estimated
intracellular concentration of Trx (10 µM) is higher
compared with a 10-fold lower one for Grx1 (30, 31) suggesting a
preference in vivo for thioredoxin as hydrogen donor for
sulfate reduction. The kinetic constants for thioredoxins and the
glutaredoxins in the reaction catalyzed by PAPS reductase are compared
in Table I.

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Fig. 2.
Initial velocity kinetics of the PAPS
reductase. A, Hanes-Woolf plot of [Trx1])
v 1 versus [Trx1] varied at
different PAPS concentrations. [PAPS] (µM): = 2.5, = 3.3, × = 5, = 10, = 16.7, = 30. The lines show a
common intersect on the y axis above the origin.
Inset, secondary plot of the slopes of the primary plot
versus [PAPS] 1 to obtain
Vmax and Km trx1.
B, Hanes-Woolf plot of [PAPS] v 1
versus [PAPS] varied at different Trx1 concentrations.
(Trx1) (µM): = 1, × = 1.3, = 2, = 6, = 20. Inset, secondary plot of the slopes of the primary plot
versus [Trx1] 1 to obtain
Vmax and Km PAPS.
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Fig. 3.
Initial velocity kinetics of the PAPS
reductase. A, Hanes-Woolf plot of [Grx1]
v 1 versus [Grx1] varied at different PAPS
concentrations. [PAPS] (µM): = 2.5, = 3.3, × = 5, = 10, = 16.7, = 30. The lines show a common intersect
with the y axis above the origin as in Fig. 2.
Inset, secondary plot of the slopes of the primary plot
versus [PAPS] 1 to obtain
Vmax and Km grx1.
B, Hanes-Woolf plot of [PAPS] v 1
versus [PAPS] varied at different Grx1 concentrations.
[Grx1] (µM): = 1, = 1.3, × = 2, = 6, = 20. Inset, secondary plots of the slopes of the primary
plots versus [Grx1] 1 to obtain
Vmax and Km PAPS.
|
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Table I
Kinetic parameters of PAPS, Trx1, Trx2, Grx1, and Grx1C14S for PAPS
reductase
The measurements were repeated three times with three enzyme
preparations for each Km value using five
concentrations of reductant versus six concentrations of
PAPS. The Km values were extracted from slope
replots of the corresponding Hanes-Woolf plots assuming a ping-pong
bireactant enzyme mechanism. The deviation is given as standard
deviation.
|
|
When Trx2 was used as reductant, Vmax was not
significantly affected, but Km increased 2.4-fold to
34.2 µM. The higher Km for Trx2 may be
seen as an indication of a slightly disturbed contact in the charge
transfer complex between enzyme and reductant. Hence, the enzyme rate
supported by Trx2 may be sufficient for heterotrophic growth, but the
low level of expression of Trx2 reported by Miranda-Vizuete et
al. (6) may be the reason why Trx1/Grx1 double mutants score as
cysteine auxotrophs. This explanation is supported with the recent
finding that overexpression of Trx2 enables a trxA grxA
double mutant to grow on minimal medium without the supplement of
cysteine (33). Miranda-Vizuete et al. (6) also described a
5-fold lower activity of Trx2 in comparison with Trx1 in the insulin
disulfide reductase assay. As pre-reduction of Trx2 with dithiothreitol
or deletion of an N-terminal extension containing two additional
CXXC motifs increased its activity considerably the authors
proposed that the two CXXC motifs present in Trx2 control
the disulfide reductase activity by their redox state. In the PAPS
reductase assay the redox state of Trx2 had no effect upon the
enzymatic activity, because pre-incubation with dithiothreitol did not
stimulate the rate of sulfite formation. Grx2 and Grx3 were both
inactive as hydrogen donors for PAPS reductase, whereas the Grx1 mutant
C14S retained 6% of the wild type activity (330 milliunits
mg
1) with a slightly increased affinity as reflected in a
lower Km of 7.2 µM. A reduction of the
homodimeric PAPS reductase by Grx1C14S seems noteworthy, because it
would lead to a possibly transient glutaredoxin enzyme-mixed disulfide
that becomes accessible to reduced glutathione. Indeed, the monothiol
mutant retained 38% of the wild-type protein in the glutathione
disulfide oxidoreductase assay but was completely inactive as hydrogen
donor for ribonucleotide reductase (19). The lack of activity observed
with Grx2 is not surprising because of its unusual structure (20).
Grx3, however, shares a 33% overall identity with Grx1 and contains
several patches of identical residues that have been identified as
indispensable for redox functions (34, 35). Moreover, the secondary and tertiary structure of both glutaredoxins were found to be very similar
(35). However, the vicinal active site cysteine residues in Grx3 are
surrounded by charged amino acids that differ significantly from the
residues in Grx1 (34). This may have an influence on the redox
potential of Grx3, which is more positive than Grx1 and on the area of
contact in hydrophobic protein-protein interactions. A positive
potential surrounding the negative charge of the thiolate of the active
site most certainly is necessary to guide the protruding end of the Grx
molecule toward its substrate buried in the catalytic site of the enzyme.
Our results give an explanation for the essential role of Trx1 or Grx1
for the growth of E. coli on minimal medium containing sulfate as the only source of sulfur (4). Because the apparent Km values of Trx1 and Grx1 are similar
(approximately 14 µM) and much higher than the
corresponding values for ribonucleotide reductase (30, 31), the sulfate
reduction pathway would seem less favored than that of ribonucleotide
reduction, which is essential for DNA synthesis. Furthermore, the even
higher apparent Km value for Trx2 and its relatively
low expression level (6) makes this new thioredoxin a poor
electron donor to PAPS reductase in particular when the full demand is
on to supply DNA replication with the essential deoxyribonucleotides.
 |
ACKNOWLEDGEMENTS |
We thank C. Svensson, EMBL, Heidelberg for
help with the laser mass spectroscopy.
 |
FOOTNOTES |
*
This study was supported financially by the Deutsche
Forschungsgemeinschaft and the Swedish Cancer Society (961).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Biochemistry of Plants,
Faculty of Biology, Ruhr-University, P.O. Box 10 21 48, 44780 Bochum,
Germany. Tel.: 49-234-7003657; Fax: 49-234-7094396; E-mail:
jens-dirk.schwenn{at}ruhr-uni-bochum.de.
 |
ABBREVIATIONS |
The abbreviations used are:
APS, adenylylsulfate;
Grx, glutaredoxin;
OAS, O-acetyl-L-serine;
PAP, adenosine
3',5'-bis-phosphate;
PAPS, 3'-phosphoadenylylsulfate;
Trx, thioredoxin;
PAGE, polyacrylamide gel electrophoresis.
 |
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