From the Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Département de Biologie Moléculaire et Structurale-Chimie Biologie Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, 17 avenue des Martyrs, 38054 Grenoble, Cedex 09, France
Received for publication, December 19, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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Anaerobic ribonucleotide reductase
provides facultative and obligate anaerobic microorganisms with the
deoxyribonucleoside triphosphates used for DNA chain elongation and
repair. In Escherichia coli, the dimeric Class III ribonucleotide reductases
(RNRs)1 are found in
anaerobic bacteria where they supply the cell with the dNTPs
needed for DNA chain elongation and repair (1). The dNTPs are obtained by direct reduction of their corresponding ribonucleotides in a
reaction basically similar for the three RNR classes and initiated by
hydrogen abstraction at the C3' ribose substrate by a cysteinyl radical (2). The cysteinyl radical itself is derived from a stable
protein radical (a tyrosyl radical in class I and a glycyl radical in
class III) or from an organometallic cofactor (class II).
In the anaerobic class III enzyme from Escherichia coli, the
protein radical is located on the polypeptide backbone at the Gly681 residue of the dimeric A second characteristic sets class III apart from the two other
classes. In class III enzymes the electrons needed for the reduction of
the ribonucleotides are provided by formate (11, 12). On the contrary,
in classes I and II, these electrons are supplied by NADPH through
thioredoxin or glutaredoxin (13).
In this paper, we show that the thioredoxin system efficiently replaces
DTT during activation of class III RNR. Our data suggest that this
system keeps the conserved cysteines of the C terminus of the Materials--
Enzymes and other components of the anaerobic
ribonucleotide reductase system have been obtained as previously
described (10, 14). Thioredoxin and thioredoxin reductase from E. coli, Chlamydomonas reinhardtii, and Arabidopsis
thaliana were provided by Dr. J. Covès (Grenoble, France)
and J. P. Jacquot (Nancy, France). AdoMet and
S-adenosylhomocysteine (SAH) were purchased from
Roche Diagnostics.
Methods--
Protein concentration was determined by the method
of Bradford (15), standardized by amino acid analyses of each different protein. UV-visible spectra were recorded inside the glove box with a
Hewlett-Packard 8453 diode array spectrophotometer coupled to the
measurement cell by optical fibers (Photonetics system).
Enzyme Assay--
In the first activating step, the Preparation of the Reduced Assay of the anaerobic reductase activity is routinely measured by
the formation of dCTP. The As shown in Fig. 1 we demonstrated that, with catalytic
thioredoxin oxidoreductase and NADPH,
thioredoxin was, at micromolar concentration, at least as efficient as
DTT in the millimolar range for enzyme activity. TRXs from different
sources (E. coli and A. thaliana) were found
equally effective (not shown). A Km(app) of
0.8 ± 0.3 µM for TRX from E. coli in
this reaction has been determined (Fig. 1, inset). Moreover
the effect of TRX on the activity was restricted to the activation step
only because there was almost no activity when TRX was added in the
reduction step along with SAH. This behavior closely resembled that
observed with DTT. No TRX-dependent activity could be
observed in the absence of TRR.
2 enzyme
contains, in its active form, a glycyl radical essential for the
reduction of the substrate. The introduction of the glycyl radical
results from the reductive cleavage of S-adenosylmethionine catalyzed by the reduced (4Fe-4S) center of a small activating protein called
. This activation reaction has long been known to
have an absolute requirement for dithiothreitol. Here, we report that
thioredoxin, along with NADPH and NADPH:thioredoxin oxidoreductase, efficiently replaces dithiothreitol and reduces an unsuspected critical
disulfide bond probably located on the C terminus of the
protein.
Activation of reduced
protein does not require dithiothreitol or
thioredoxin anymore, and activation rates are much faster than
previously reported. Thus, in E. coli, thioredoxin has very
different roles for class I ribonucleotide reductase where it is
required for the substrate turnover and class III ribonucleotide
reductase where it acts only for the activation of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 (2 × 80 kDa)
protein (3). The glycyl radical (Gly·) is formed by the
concerted action of the following four components: (i) a reducing
system consisting of NADPH, flavodoxin, and NADPH:flavodoxin reductase
(4, 5); (ii) a 17.5-kDa iron-sulfur protein called
or
"activase" (6, 7) whose function is to catalyze the reductive
cleavage of (iii) an acceptor molecule identified as S-adenosylmethionine (AdoMet) (8, 9). The reaction also requires (iv) dithiothreitol (DTT), a nonphysiological reductant (10).
In the inactive resting state, the two proteins
and
forms a
tight
2
2 complex, but under the reducing conditions leading to
the introduction of the radical (the activation reaction), the small
protein is able to activate several molecules of the
protein
(6, 7).
polypeptide in a reduced form needed for radical generation. This
result solves an intriguing question concerning the function of DTT and
the identity of its unknown physiological counterpart.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein
(1.2 µg), in a total volume of 35 µl, was incubated on a manifold
for 60 min at room temperature under a flux of moist argon with DTT
(0.5-8 mM) or TRX (0.5-10 µM) and the
activation mix containing 55 mM Tris-HCl, pH
8.0, 55 mM KCl, 3.4 mM NADPH, 130 µM AdoMet, 0.1 µg of the
protein, 0.5 µM flavodoxin, 0.15 µM flavodoxin
oxidoreductase, and 0.15 µM thioredoxin reductase. The
latter was omitted in the standard (DTT) assay. In the second step, 15 µl of the substrate mixture (giving a final concentration of 1.4 mM [3H]CTP (20-30 cpm/pmol), 1 mM ATP, 10 mM MgCl2, 10 mM HCOONa, and 100 µM EDTA) was added
to initiate the reduction of the substrate. When no interference from
further activation was desired, 200 µM SAH (final
concentration) was included in the substrate mix. The reaction was
stopped after 20 min by opening the tubes to air and addition of 0.5 ml
of 1 M HClO4. The solution was then worked up as described earlier (14). One unit of enzyme activity is
defined as the formation of 1 nmol of dCTP per min.
Protein--
The
protein (820 µM) was incubated with TRX (5.7 µM), TRR
(1.3 µM), and NADPH (1.6 mM) in 0.1 M Tris-HCl, 50 mM KCl, pH 8.0. Oxidation
of NADPH was monitored at 340 nm inside the glove box by UV-visible
spectroscopy. After 2 h, the solution was diluted 20-fold with
buffer and loaded on a dATP-Sepharose affinity column (2 ml) at 0.2 ml·min
1. The column was washed with 10 ml of buffer,
and the reduced
protein was eluted with buffer containing ATP (1 mM). The reduced
protein was then repetitively
concentrated with an ATP-free buffer to a final concentration of 10-20
mg·ml
1 by ultrafiltration (NanoSep 30K; Filtron Corp.)
inside the box and was then frozen. Alternatively, the reduced
protein was prepared inside the box by overnight incubation of the
as-isolated form (1 mM) with 100 mM DTT
followed by Sephadex G-25 chromatography over a long (80-ml) column.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein is first activated under
anaerobic conditions by a 1-h incubation with a source of electrons
(the flavodoxin system or chemical reductants), AdoMet, and the
activating
protein (6). This step is absolutely dependent on the
presence of small thiol molecules, the most effective being DTT (10).
In a second step, the reduction step, the activated enzyme is incubated
with the CTP substrate, the positive effector ATP, Mg2+,
and formate. SAH can be added to this step to allow for the reduction
to proceed in the absence of further activation (16). This standard
assay was here modified by addition of 100 µM EDTA in the
buffer as this greatly stimulated the
activity.2
View larger version (19K):
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Fig. 1.
DTT and TRX concentration dependence of CTP
reductase activity. The protein (1.25 µg) was incubated
anaerobically for 1 h at room temperature with the activation mix
as described under "Experimental Procedures" in the absence (
)
or in the presence of DTT (
) or TRX (
). The tubes containing the
protein
alone (
) were then supplemented with TRX just
before adding the substrate mix containing SAH (200 µM)
to all tubes. Inset, Lineweaver-Burk plot with varying
concentrations of TRX. The experimental values were fitted to a linear
regression program giving a correlation factor r = 0.997 (Kaleidagraph).
In Fig. 2 is shown the
time-dependent decrease of the absorbance of NADPH (1.6 mM) at 340 nm when a solution of the protein (820 µM) was complemented inside the glove box first with TRR (1.3 µM) and after 10 min with TRX (5.7 µM). Control experiments done without protein
showed
a negligible decrease of the absorbance at 340 nm compared with that
seen in Fig. 2. Quantitative analysis of these data showed that the
reaction was almost completed after 90 min when about 820 µM of NADPH has been converted to NADP+. This
amount exactly matched the concentration of the
polypeptide. This
indicated that protein
enjoyed a 2-electron reduction, and because
this reaction is catalyzed by TRX it also strongly suggested that, in
the as-isolated form, protein
carries a disulfide group amenable to
reduction. Taken together these experiments suggest that the activation
of the anaerobic ribonucleotide reductase is dependent on the presence
of cysteine-free SH groups on protein
.
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Reduced protein , after treatment with the TRX system as depicted in
Fig. 2, was isolated by chromatography on an affinity dATP-Sepharose
column inside the glove box. After washing the column with 5 volumes of
buffer, the reduced protein was eluted with the admission of 1 mM ATP in buffer. It was then assayed for its CTP reductase
activity. In Fig. 3 is compared the
activity of the reduced protein
isolated after dATP-Sepharose with
that of the as-isolated (oxidized) protein
in the presence or in the absence of DTT. The activity was linearly correlated with the
amount of enzyme except for the data at high protein concentration, which may be explained by the near exhaustion of the substrate. In
agreement with previous data, the oxidized
protein alone was found
completely inactive and required DTT for activity (10). On the other
hand, the reduced
protein was found active in the absence of any
additional reductant and displayed the highest enzyme activity. The
same results essentially were obtained when protein
was treated
with DTT and isolated by Sephadex G-25 chromatography under strict
anaerobic conditions (data not shown). However, in that case, reduction
of protein
required a high concentration of reductant (100 mM) and long incubation time.
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Our data show that reduction of protein either by DTT or TRX is a
rather slow process. Accordingly, in previous studies using DTT and
oxidized preparations of protein
, activation of the enzyme required
prolonged incubation (at least 30-45 min) (8, 9, 14). However, as
shown in Fig. 4, using reduced protein
in the absence of DTT or TRX, activation occurred at a much faster
rate, because about 50% of the maximal activity was achieved in less
than 1 min. It is thus very likely that in the previously reported
studies, enzyme activation was in fact rate-limited by reduction of
protein
.
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DISCUSSION |
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Cysteines are central to the activity of class I and II ribonucleotide reductases (2). They participate either as a thiyl radical for initiating substrate reduction or as a dithiol in the subsequent steps leading to the deoxyribose product (2, 17). The latter cysteine pair is maintained in the reduced form by thioredoxin or glutaredoxin (13). In class III RNR, alignments of the 20 nrdD sequences presently available have revealed, among the 15-20 Cys generally present, the occurrence of five invariants, one highly conserved and three moderately conserved cysteine residues (18). Site-directed mutagenesis studies done on the enzyme from the bacteriophage T4 have demonstrated that two of these invariants are directly involved in the turnover of the reaction in agreement with the presence of two cysteines in the substrate site observed in the three-dimensional structure of the protein (12, 19). The three other invariants are part of a CXXCX14CXXC motif (not visible in the three-dimensional structure) located in the C terminus of the polypeptide. Each of these cysteines was essential for the formation of Gly· and was thus proposed to participate to radical transfer reactions during enzyme activation (19).
Activation of class III RNR has long been known to be a slow process
and to rely upon the obligate presence of DTT in the activation mix
(14). However, nothing was known about the function of DTT or the
nature of its physiological counterpart. The data reported here
strongly suggest that the role of DTT was to reduce an unsuspected
disulfide on protein . From the study of Andersson et
al. (19), it is likely that this disulfide, in the E. coli enzyme, is located on the Cys662/665 pair, but
this has to be confirmed with appropriate mutants. This disulfide can
be reduced by the TRX/TRR system resulting in a fully active enzyme. As
a consequence, reduced protein
can now be activated in a fast
reaction that does not require DTT or the TRX/TRR system anymore.
It thus seems that, in previous studies with the oxidized preparations,
protein activation not only resided in the introduction of the
glycyl radical but also implicated the reduction of important cysteines
by DTT. The fact that the activation reaction becomes much faster with
reduced protein
(Fig. 4) indicates that the rate-limiting step
during DTT-dependent activation reaction was not the
generation of Gly· by itself but instead the reduction of
protein
.
At this stage it is difficult to know the exact function of the thiols
in the C terminus of protein . They may have a structural role in
the binding of protein
and/or AdoMet. They also may be involved in
a radical chain transfer from AdoMet, supposed to bind at the interface
of the two proteins
and
, to the glycine residue on protein
as proposed by Andersson et al. (19). Finally they could be
directly involved in the cleavage of AdoMet. Our recent finding that
the latter reaction is triggered by addition of DTT to the
(4Fe-4S)+/AdoMet complex on one hand and that DTT binds to
the cluster3 in the
other makes this third alternative very attractive (8).
It is well established now that very few cystine pairs survive the
reducing conditions existing inside the anaerobic cell (20, 21). In
addition to the redoxin systems, E. coli contains a high
concentration of GSH, itself able to sustain CTP reductase activity
in vitro (not shown). So, in vivo, protein is
likely to have all of its cysteines in their reduced form providing
conditions for a rapid activation. The requirement for DTT or TRX
in vitro might just be a consequence of the isolation and
oxygen sensitivity of the protein. The study of the activation reaction
conducted with reduced protein
is expected to solve many of the
questions still unanswered.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Covès (Grenoble, France) and J. P. Jacquot (Nancy, France) for graciously providing pure recombinant thioredoxin and thioredoxin reductase from E. coli, C. reinhardtii, and A. thaliana.
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FOOTNOTES |
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* 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 may be addressed. Tel.: 33 04 76 88 91 07;
Fax: 33 04 76 88 91 24; E-mail: emulliez@cea.fr.
§ To whom correspondence may be addressed. Tel.: 33 04 46 88 91 03; Fax: 33 04 76 88 91 24; E-mail: mfontecave@cea.fr.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.C000895200
2 Unpublished results.
3 Unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: RNR(s), ribonucleotide reductase(s); TRX, thioredoxin; TRR, thioredoxin reductase; AdoMet, S-adenosylmethionine; DTT, dithiothreitol; SAH, S-adenosylhomocysteine.
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