From the Department of Biochemistry 1, Medical Nobel
Institute, MBB, Karolinska Institute, S-17177 Stockholm, Sweden and the
§ Department of Genetics and Microbiology, Faculty of
Sciences, Autonomous University of Barcelona, Bellaterra,
E-08193 Barcelona, Spain
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ABSTRACT |
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Three separate classes of ribonucleotide
reductases are known, each with a distinct protein structure. One
common feature of all enzymes is that a single protein generates each
of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding
effectors (ATP or various deoxyribonucleoside triphosphates) that
direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this
site inhibits the activity of these enzymes. X-ray crystallography has
localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one
from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and
Thermotoga maritima). Each enzyme has an allosteric site
that binds ATP or various deoxyribonucleoside triphosphates and that
regulates its substrate specificity according to the same rules as for
class I and III enzymes. dATP does not inhibit enzyme activity,
suggesting the absence of a second active allosteric site. For the
L. leichmannii and T. maritima enzymes, binding
experiments also indicate the presence of only one allosteric site.
Their primary sequences suggest that these enzymes lack the structural
requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second
site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its
function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the
structural basis for the site. Also a large subgroup (Ib) of class I
enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may
have arisen by divergent evolution from class III enzymes.
Among the fascinating properties of ribonucleotide reductases is
their allosteric regulation of substrate specificity (general review in
Ref. 1). These enzymes balance the reduction of ribonucleotides in a
way that satisfies the cell's need for all four building blocks
required for DNA synthesis. One protein catalyzes four separate
reactions. The substrate specificity of the catalytic site for a given
ribonucleotide is determined by binding of a specific
deoxyribonucleoside triphosphate
(dNTP)1 or ATP to an
allosteric site (substrate specificity site). Thus, binding
of ATP or dATP induces activity toward pyrimidine ribonucleotides; binding of dTTP induces activity toward guanine ribonucleotides; and
binding of dGTP induces activity toward adenine ribonucleotides. These
effects are largely the same for all ribonucleotide reductases studied
so far, except for some viral enzymes (2). Many reductases have, in
addition, a second allosteric site (activity site) that regulates their overall activity, with ATP promoting and dATP inhibiting enzyme activity (1).
Three classes of ribonucleotide reductases occur in nature. In addition
to the earlier mentioned allosteric regulation of substrate
specificity, all enzymes share a similar common free radical mechanism
for the reduction of ribose and contain, for this purpose, a free
radical as part of their protein structure. The three classes differ in
the way in which the protein radical is produced and have evolved
distinct protein structures (1, 3-5).
Class I reductases, with the aerobic Escherichia coli enzyme
as the prototype, contain a tyrosyl free radical and have an The signum of class II reductases (NrdJ proteins) is their dependence
on adenosylcobalamin, which acts as a radical generator and thus
supplies the same function as the R2 protein of class I reductases. All
class II reductases contain a single polypeptide chain, functionally
related to the R1 protein of class I enzymes. They are widely spread
among aerobic and anaerobic bacteria and do not depend on oxygen. The
enzyme from Lactobacillus leichmannii (9-11) was, for a
long time, the only well characterized member of this class and became
its prototype. When, in recent years, the amino acid sequences of many
more class II enzymes were determined, it became apparent that the
L. leichmannii enzyme has an unusual sequence, different
from those of most other class II as well as class I and III enzymes.
Class III reductases (NrdDG proteins), with the anaerobic E. coli reductase as a prototype (12), have again an
Within one class, the large The allosteric properties of several class I reductases (16, 17) and
one class III reductase (18) have been investigated in detail by
kinetic and effector binding experiments. Our knowledge of the
allosteric behavior of class II enzymes is, however, incomplete. We
know next to nothing concerning the recently described archaeal and
deeply rooted eubacterial enzymes, and early studies of the L. leichmannii enzyme (19, 20) need to be complemented in the light
of recent knowledge. Here, we describe our experiments with three class
II reductases: the previous prototype enzyme from L. leichmannii (9, 10), the archaeal thermophilic enzyme from
Thermoplasma acidophilum (21), and the eubacterial
hyperthermophilic enzyme from Thermotoga maritima (14). Both
T. acidophilum and T. maritima are deeply rooted
organisms. L. leichmannii, instead, is a highly specialized
microorganism, adapted to life in milk. The three enzymes had the
opportunity to diverge extensively during evolution. Despite this,
their substrate specificity was regulated in a similar manner,
according to the same rules as for class I and III reductases. The
thermophilic organisms showed a clear allosteric regulation only at
elevated temperatures, suggesting that the proteins at lower
temperature are not sufficiently flexible to transmit the
conformational change resulting from effector binding to the catalytic
site. None of the three enzymes was inhibited by dATP, and they all
thus lack a functional activity site. However, the T. acidophilum reductase (but not the other two enzymes) meets the
structural requirements for an activity site, and in effect, it did
bind a second dATP. Our results will be discussed in light of a
previously suggested model for the evolution of ribonucleotide reduction.
Materials
Bacterial strains overproducing the L. leichmannii
(10) and T. acidophilum (21) enzymes were kindly provided by
Dr. J. Stubbe. The T. maritima ribonucleotide reductase was
purified from an overproducing E. coli strain prepared
earlier in this laboratory (14). Labeled nucleoside di- and
triphosphates were obtained from Amersham Pharmacia Biotech. They were
diluted to the following specific activities: 14C-labeled
substrates, 3-6 cpm/pmol; 3H-labeled substrates, 10-20
cpm/pmol; and 3H-labeled effectors, 200-450 cpm/pmol.
Their radiopurity was checked by chromatography on polyethyleneimine
strips. If necessary, they were purified by chromatography on
DEAE-Sephadex with a volatile buffer. Small portions of the dissolved
labeled nucleotides were stored at Purification of Ribonucleotide Reductases
Common Procedures--
Batch cultures of bacteria were grown at
37 °C in Luria-Bertani broth with 100 µg/ml ampicillin to a final
A600 of 0.5, at which point
isopropyl- Further Purification of the L. leichmannii
Reductase--
Overproduction of the enzyme was very efficient, and at
this stage, we had a total of 83 mg of protein with a specific activity of 490 units/mg of protein. Half of this protein, dissolved in 2.6 ml
of buffer A, was adsorbed onto a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) and eluted with a 0-1 M KCl gradient in
buffer A. A peak of activity was recovered at 0.23 M KCl,
and the central portion of this peak (16.5 mg of protein,
specific activity = 750 units/mg of protein) was used for
our experiments.
Further Purification of the T. acidophilum Reductase--
After
the ammonium sulfate precipitation of the protein from an extract of
20 g of bacteria, we recovered 1.0 g of protein with a
specific activity of 14 units/mg of protein. The solution was heated at
55 °C for 30 min and centrifuged, and the clear supernatant solution
was precipitated with solid ammonium sulfate to 50% saturation. The
centrifuged precipitate was dissolved in 2 ml of buffer B (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM DTT)
and freed from ammonium sulfate by dialysis against the same buffer.
The enzyme (144 mg) now had a specific activity of 46 units/mg of
protein. It was adsorbed onto a Mono Q HR column and chromatographed
with a 0-1 M KCl gradient. A peak of activity (8 mg,
specific activity = 186 units/mg of protein) appeared around 0.2 M KCl. After concentration in Centricon 30 tubes, the
protein was adsorbed onto a 10-ml column of hydroxylapatite equilibrated with 50 mM Tris-HCl, pH 8.0. Inactive protein
was removed by elution with 75 mM phosphate, pH 7.5, followed by elution of the active protein (1.7 mg, specific
activity = 246 units/mg of protein) with 200 mM
phosphate, pH 7.5. After concentration in Centricon 30 tubes, the
protein was equilibrated with 0.1 M KCl, 50 mM
Tris-HCl, pH 7.5, and 10% glycerol on a 5-ml column of Sephadex G-25,
giving a final yield of 0.51 mg of pure enzyme. The protein was highly
"sticky," and recovery was low. It was also prepared in pure form,
but again in low yield, by affinity chromatography on dATP-Sepharose as
a final purification step instead of hydroxylapatite in a procedure
related to the one described below for the T. maritima
reductase. Both preparations gave a single band on denaturing gels and,
when used in binding experiments, gave identical results.
Further Purification of the T. maritima Reductase--
After
ammonium sulfate precipitation, the protein in buffer B (1.44 g, 10 mg/ml, specific activity = 70 units/mg of protein) was heated for
10 min at 80 °C, and the resulting precipitate was removed by high
speed centrifugation. After addition of 1.15 ml of 1 M
CaCl2, the solution (114 ml, 1 mg of protein/ml, specific activity = 550 units/mg of protein) was adsorbed onto a 25-ml column of dATP-Sepharose equilibrated with 0.1 M KCl, 30 mM Tris-HCl, pH 7.5, 10 mM CaCl2,
and 2 mM DTT. The column was washed with 40 ml of the same
buffer, and the reductase was eluted with 0.5 M ammonia.
The combined fractions containing 29 mg of protein were neutralized
with 2 M NaH2PO4, and the protein
was precipitated overnight after addition of solid ammonium sulfate to
70% saturation. Denaturing gel electrophoresis at this stage showed
only two protein bands with mobilities of ~70 and 90 kDa. The
proteins corresponding to these bands could be separated by
chromatography on a column of Superdex 200 (Amersham Pharmacia Biotech)
equilibrated with 0.1 M KCl, 30 mM Tris-HCl, pH
7.5, and 2 mM DTT. The ammonium sulfate precipitate was
dissolved in 1.5 ml of this buffer and added to the column. Elution
with the same buffer resulted in the appearance of two cleanly
separated symmetrical protein peaks with mobilities corresponding to
~500 and 150 kDa, named TM1 and TM2, both enzymatically active. The
materials corresponding to each peak were concentrated in Centriprep 30 tubes. TM1 (8.5 mg, specific activity = 1500 units/mg of protein)
corresponded to the 90-kDa band on denaturing gels. Mass spectrometry
showed a single peak with a mass of 93.5 kDa.2 TM2 (2.9 mg, specific
activity = 1800 units/mg of protein) corresponded to the 70-kDa
band. Both TM1 and TM2 had the same N-terminal sequence as the enzyme
isolated from T. maritima,2 suggesting that the
smaller size of TM2 was due to C-terminal processing of the enzyme.
Assay of Reductase Activity
Under standard conditions, all three enzymes were incubated in a
final volume of 50 µl for 20 min in the presence of 0.5 mM [3H]CTP (L. leichmannii
reductase) or [3H]CDP (T. acidophilum and
T. maritima reductases), 100 mM DTT, 15 µM adenosylcobalamin, 30 mM Tris-HCl, pH 8.0, 100 µM dATP, and 10 mM CaCl2
(L. leichmannii enzyme), 10 mM MgCl2
(T. maritima enzyme), or 40 mM MgCl2
(T. acidophilum enzyme) at 37 °C (L. leichmannii enzyme), 80 °C (T. maritima enzyme), or
55 °C (T. acidophilum enzyme). The reaction was
terminated with 1 ml of ice-cold 1 M HClO4, and
the amount of [3H]dCMP formed was determined by
chromatography on Dowex-50 after 10 min of hydrolysis at 100 °C
(22). Reduction of ADP (ATP) or GDP (GTP) was assayed with
14C-labeled substrates under identical incubation
conditions except for the allosteric effector. After incubation, the
nucleotides were transformed to nucleosides by digestion with alkaline
phosphatase, and the labeled deoxyribosides were separated from the
ribosides on boronate columns (23). One unit of activity is defined as 1 nmol of product formed during 1 min. Specific activity is units/mg of
protein. Protein was determined colorimetrically (24) with crystalline
bovine serum albumin as the standard.
Sucrose Gradient Centrifugations
5-20% linear sucrose gradients in a total volume of 4.6 ml
were prepared in 50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, and 2 mM DTT. In some experiments, 15 µM adenosylcobalamin and/or 100 µM dATP was
present throughout the gradient. Solutions (0.2 ml) of the enzymes
containing catalase as an internal marker
(s20,w = 11.4 S) were layered onto the
gradient and centrifuged in a Beckman SW 50 rotor at 32,000 rpm for
18 h at 20 °C. After the run, the tubes were punctured at the
bottom, and a total of 33-38 fractions were collected and analyzed for
protein content and catalase activity. The sedimentation coefficients
of the proteins were calculated from their positions in the gradient
relative to the catalase marker.
Nucleotide Binding Experiments
The method of Ormö and Sjöberg (25) was used at
+4 °C as described previously (17, 18). No dephosphorylation of any nucleotide occurred during the course of the experiments as determined by polyethyleneimine chromatography.
Purity and Oligomeric State of the Enzymes--
Enzyme
purification as described under "Experimental Procedures" provided
large amounts of L. leichmannii and T. maritima reductases, but only limited amounts of the T. acidophilum
enzyme. All three enzymes gave rise to a single band on denaturing gel electrophoresis (Fig. 1) with positions
in accordance with their molecular masses of 82, 94, and 97 kDa for the
enzymes from L. leichmannii (10), T. maritima
(14), and T. acidophilum (21), respectively, calculated from
their amino acid compositions. In the case of the T. maritima reductase, we separated two homogeneous highly active
enzymes with different molecular masses: one with a mass of 94 kDa
corresponding to the enzyme prepared from T. maritima and a
second shorter protein of ~70 kDa that had apparently arisen
through C-terminal processing of the larger protein in E. coli. The large protein was used in our experiments; the
properties of the shorter one will be described elsewhere.2
The specific activities of the pure L. leichmannii and
T. acidophilum enzymes differed from earlier reported
values. The L. leichmannii reductase showed only half the
activity; the T. acidophilum reductase was 10 times more
active. We ascribe this to differences in assay conditions. The
reductant in our assays was DTT, whereas thioredoxin from E. coli was employed earlier with the L. leichmannii
reductase. In the case of the T. acidophilum reductase, we
optimized both the DTT and cation concentrations better than was done
in previous work.
Extensive experiments from two separate laboratories (9, 20) indicated
that the native L. leichmannii enzyme is a monomer with a
molecular mass close to 80 kDa. Also the native T. acidophilum enzyme was briefly stated to be a monomer (21),
whereas the T. maritima enzyme behaved as a high molecular
mass oligomer (14). As the oligomeric state of the reductases is
important for an understanding of the allosteric effects, we decided to
re-determine this parameter. To this purpose, we measured the
sedimentation of the enzymes in sucrose gradients under various
conditions that might affect their aggregation, including different
combinations of adenosylcobalamin, dATP, DTT, and Mg2+. We
had used sucrose gradient centrifugation with advantage earlier to
determine the oligomeric structures of class I (26) and III (13)
ribonucleotide reductases. Gradients were standardized with the R1E
protein of Salmonella typhimurium (161 kDa,
s20,w = 7.7-7.9 S), aldolase (158 kDa,
7.9 S), and bovine serum albumin (67 kDa, 4.8 S). Irrespective of
conditions, the L. leichmannii enzyme (monomer = 82 kDa) sedimented at 5.6-5.7 S, the T. acidophilum enzyme
(monomer = 97 kDa) at 8.4-8.6 S, and the T. maritima
enzyme (monomer = 94 kDa) at 10.2 S. All runs were made at room
temperature, but the T. maritima enzyme was heated to
80 °C for 10 min in the presence of 20 µM
adenosylcobalamin, 2 mM DTT, 0.1 mM dATP, and 10 mM MgCl2 before centrifugation. The
sedimentation coefficients of the three reductases suggest that, in
solution, the L. leichmannii enzyme is a monomer, the
T. acidophilum enzyme a dimer, and the T. maritima enzyme a higher oligomer. As the T. maritima
enzyme normally operates above 80 °C and centrifugations were at
room temperature, more extensive studies are required to establish its
true oligomeric state.
General Properties of the Enzyme Reactions--
Our aim was to
study the influence of allosteric modulators on the substrate
specificity and activity of the reductases under optimal assay
conditions. We first optimized the reduction of CDP (CTP) with each
enzyme and then used the results to study effector requirements for the
reduction of purine ribonucleotides. As reported earlier (9, 14, 21),
the activity of each of the three enzymes is completely dependent on
the presence of adenosylcobalamin and high concentrations (100 mM) of DTT. Substrates for the reaction are ribonucleoside
diphosphates for the T. acidophilum and T. maritima reductases (14, 21) and triphosphates for the
L. leichmannii enzyme (9). Mg2+ and
Ca2+ ions stimulated all enzymes in the presence of
allosteric effectors, but were not absolutely required. The T. maritima enzyme worked equally well with both ions, whereas
Ca2+ was slightly more active with the L. leichmannii reductase and Mg2+ with the T. acidophilum enzyme. In the latter case, the optimal concentration
was as high as 40 mM, whereas the other two enzymes showed
the highest activity at 10 mM. In the absence of allosteric effectors, both ions were inhibitory. These results were obtained in
comprehensive experiments with CDP (CTP) as substrate, but were also
verified for purine ribonucleotides in some experiments (data not
shown). With guanine nucleotides, experiments with Ca2+ are
limited to concentrations below 5 mM, as precipitation
occurs at higher values. As reported earlier, temperature optima were 55 °C for the T. acidophilum enzyme (21) and
90 °C for the T. maritima enzyme (14).
Allosteric Properties of Reductases--
A summary of the
modulation of the substrate specificity of all three reductases by
nucleoside triphosphates is given in Table I. Table I provides selected results from
extensive experiments with each reductase in which we determined the
activity with each substrate at various modulator concentrations. The
effects were strikingly similar to those observed earlier with class Ib
enzymes. Active effectors were ATP and the four dNTPs. Under optimal
conditions and in the presence of Mg2+ or Ca2+,
the following general pattern emerges for all three enzymes: ATP and
dATP stimulated reduction of CDP (CTP); dGTP stimulated reduction of
ADP (ATP); and dTTP stimulated reduction of GDP (GTP). With the
T. acidophilum enzyme, GDP reduction was also strongly stimulated by dCTP. This is an unusual effect for dCTP. In most cases,
a 5-10-fold increase in activity was seen. dATP at concentrations up
to 1 mM did not inhibit CDP (CTP) reduction, in contrast to what occurs with class Ia and III enzymes. In the absence of
Mg2+ or Ca2+, the effects were smaller. Then,
the reaction both proceeded faster in the absence of effector and had a
lower rate in its presence (data not shown). With the T. acidophilum enzyme, temperature was also an important parameter,
as found earlier for the T. maritima enzyme (14). A similar
result is now shown for the T. acidophilum reductase (Fig.
2). In repeated experiments, CDP
reduction was stimulated by both ATP and dATP at 60 °C, but not at
40 °C. Also the reductions of ADP and GDP by the two enzymes showed
a more pronounced stimulation at elevated temperatures (data not
shown). With the L. leichmannii enzyme, there was no
difference between 25 and 37 °C.
The E. coli aerobic ribonucleotide reductase, a class Ia
enzyme, loses its requirement for Mg2+ and allosteric
effectors if supplied with high concentrations of sodium acetate (27).
We wished to find out if a cobalamin-dependent enzyme
behaved similarly. With CTP as substrate, the L. leichmannii enzyme had a specific activity of 84 units/mg of protein without effector, 674 units/mg of protein with dATP and Ca2+, and
414 units/mg of protein with 0.4 M sodium acetate. Addition of either dATP or Ca2+ (or both) decreased the activity of
the sodium acetate-stimulated reaction. Similar results were obtained
for GTP, whereas ATP reduction was not affected. These data show that
also a B12 enzyme could replace allosteric effectors with
high concentrations of sodium acetate.
Binding Experiments with the L. leichmannii Enzyme--
Binding of
isotopically labeled dATP, dGTP, or dTTP is shown in the form of
Scatchard plots in Fig. 3A.
The abscissa gives the molecules of ligand bound per
polypeptide chain at increasing concentrations of the nucleotide. The
number of total binding sites is obtained by extrapolation. The
dissociation constants (KD) for each ligand can be
calculated from the slopes of the curves. Deviations from linearity
indicate heterogeneity of sites. For the L. leichmannii
reductase, linear curves were obtained with all three dNTPs,
extrapolating to values close to 1, suggesting binding of one molecule
of each ligand/polypeptide to a single site. The slopes give
KD values of 2 µM (dATP), 3 µM (dGTP), and 4 µM (dTTP). The results are
from experiments with 10 mM Mg2+. In the
presence of 10 mM Ca2+ in place of
Mg2+, dATP was bound linearly with a lower
KD of 0.4 µM (data not shown).
To determine whether a single or several different sites bound the
various ligands, we tested competition of unlabeled nucleotides with
binding of labeled dATP. Increasing amounts of the unlabeled nucleotides were tested together with a fixed concentration of 10 µM dATP (Fig. 3B). dCTP, dGTP, and dTTP at
high enough concentrations completely prevented dATP from binding, with
dGTP being most and dCTP least efficient. In contrast, up to 2 mM ATP had little effect. The data agree with a single
binding site for the four dNTPs. We believe that the apparent lack of
competition by ATP depends on the high concentration of dATP required
for saturation of the enzyme. ATP is an efficient effector for CTP
reduction (cf. Table I).
Binding Experiments with the T. maritima Reductase--
This
enzyme bound both dATP and dGTP with a much higher affinity than the
L. leichmannii reductase, with KD values of 0.1 and 0.3 µM, respectively (Fig.
4A). The binding stoichiometry was again one molecule of dATP or dGTP/polypeptide chain, and binding
was linear. In competition experiments, dCTP, dGTP, and dTTP as well as
ATP blocked binding of dATP (Fig. 4B). Of the dNTPs, dGTP
appeared most and dCTP least effective in this respect, similar to the
situation with the L. leichmannii reductase. Now also ATP
wiped out dATP binding. Due to the high affinity of the enzyme for
effectors, dATP was used at a 30-fold lower concentration than in the
L. leichmannii experiment. The experiment suggests that the
T. maritima reductase contains a single binding site for all
tested nucleotides. In an additional experiment, competition between
dATP and dGTP was studied quantitatively by measuring the binding of
increasing amounts of labeled dATP in the presence of two fixed
concentrations of dGTP. The results of such an experiment with 1.5 and
3.0 µM dGTP, respectively, are shown in the Scatchard plots of Fig. 4C. Also in the presence of dGTP, dATP binding
extrapolates to one binding site, but now with higher apparent
dissociation constants. Inhibitor constants for dGTP of 0.3 and 0.5 µM, respectively, can be calculated, in good agreement
with the KD of 0.3 µM for dGTP found
in the experiment of Fig. 3A, providing convincing evidence
for binding of dATP and dGTP to the same site of the enzyme.
Binding Experiments with the T. acidophilum Reductase--
The
results obtained with the T. acidophilum reductase differed
in some important aspects from those with the two previous enzymes.
This reductase is a homodimer, and binding stoichiometries will be
given per enzyme dimer and not, as for the two previous enzymes, per
polypeptide. One dimer of the T. acidophilum reductase bound
two molecules of dGTP and four molecules of dATP (Fig.
5A). Thus, the binding
stoichiometry of the T. acidophilum reductase was identical
to that of the E. coli R1 dimer that also has the capacity
to bind two molecules of dGTP and four molecules of dATP. Of these, two
molecules of dGTP or two molecules of dATP bind to the substrate
specificity sites, and two additional molecules of dATP bound to the
activity sites. However, whereas binding of dATP to the activity sites
of R1 inhibits ribonucleotide reduction, the T. acidophilum
reductase was not inhibited similarly (cf. Table I). The
enzyme bound both dGTP and dATP linearly with high affinity
(KD for dGTP = 0.04 µM and for
dATP = 0.03 µM). The linearity of dATP binding shows
that the nucleotide was bound to each of the four sites with equal
affinity. This differs from the E. coli R1 protein, where
the specificity sites have a considerably higher affinity for dATP than
the activity sites.
We next tested competition by increasing amounts of unlabeled
nucleoside triphosphates for binding of dATP at a fixed concentration of dATP that approached saturation (Fig. 5B). ATP could
block dATP binding almost completely, whereas dTTP and dCTP decreased dATP binding only to at most 50%, suggesting that dATP and ATP share
all four sites, but that dTTP and dCTP access only two of them. The
results with dGTP were less clear. In Fig. 5B as well as in
two additional experiments (data not shown), dGTP decreased dATP
binding to between 30 and 40%, values that did not change appreciably
at high dGTP concentrations. To further study competition with dGTP, we
determined binding curves for dATP at either 7.5, 50 or 200 µM dGTP (Fig. 5C) in an experiment similar to
the one depicted for the T. maritima reductase in Fig.
4C. In the latter case, there was clear competition between
dATP and dGTP for a single site on the enzyme. With the T. acidophilum reductase, the Scatchard plot for dATP binding is
concave at 50 and 200 µM dGTP and extrapolates to two
sites/dimer, as compared with four sites without competitor (Fig.
5C). Identical curves were obtained at the two dGTP
concentrations. The data suggest that only two of the four sites were
available for dATP binding when dGTP was bound to the enzyme. Once dGTP
had occupied its two sites, further addition of dGTP had no effect. The
curvature of the binding curve for dATP in the presence of dGTP
indicates that the two sites remaining for dATP binding are
heterogeneous and bind dATP with different affinities. This then may
explain why dGTP in the experiment of Fig. 5B apparently
competes with >50% of dATP: when two of the four sites of the dimer
are occupied by dGTP, the affinity of one of the sites remaining free
for dATP binding decreases, resulting in lower occupancy by dATP at the
given concentration of the ligand.
In conclusion, the binding data with the T. acidophilum
reductase suggest that the dimer contains a total of four binding sites
for dATP and ATP, two of which also have the ability to bind dCTP,
dGTP, and dTTP. This differs from the other two B12 enzymes
that contain a single type of allosteric site capable of binding all
dNTPs and ATP, but resembles the E. coli R1 protein, a class
Ia reductase. On the other hand, the catalytic behavior of the T. acidophilum reductase resembles that of the other B12 enzymes and differs from that of the E. coli R1 protein in
that the T. acidophilum enzyme is not inhibited by dATP.
The "textbook" model for the allosteric regulation of
ribonucleotide reduction is largely relevant for class Ia enzymes,
including mammalian reductases. It has its origin in early effector
binding experiments with the R1 protein of the E. coli
reductase (16) and postulates the existence of two types of allosteric
sites, two each on one R1 homodimer (or one each per polypeptide chain, as shown in Fig. 6). One type regulates
the substrate specificity of the enzyme, as outlined in the
Introduction, by binding ATP, dATP, dGTP, or dTTP; the other regulates
overall activity by binding ATP or dATP (Fig. 6). Binding of effectors
results in conformational changes that are transmitted to the active
site and there affect the catalytic process. The two allosteric sites
interact and operate in concert. This general model has withstood major
challenges during the 30 years of its existence. It has received strong
structural support from the recent x-ray data of complexes between
effectors and the E. coli R1 protein (8).
INTRODUCTION
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Abstract
Introduction
References
2
2-structure (6). The large
2-dimer has been named the R1 protein (NrdA), and the
small
2-dimer has been named the R2 protein (NrdB). The
tyrosyl radical forms part of the R2 polypeptide, which also contains
an oxygen-linked diferric center required for radical generation. This
process requires oxygen, and class I enzymes function therefore only in
aerobic organisms, both bacteria and eukaryotes. The R1 protein binds
substrates and allosteric effectors and is the catalytic part of the
enzyme. Class I has been divided into two subgroups (Ia (NrdAB) and Ib
(NrdEF)) that differ from each other functionally and in their primary
structures (7). In contrast to Ia, the class Ib reductases contain no allosteric activity site. The three-dimensional structures of complexes
between the allosteric effector dTTP (specificity site) or the effector
analog AMPPNP (activity site) and the R1 protein of the E. coli class Ia reductase have been solved, and the amino acid
residues involved in the binding of each effector were identified (8).
2
2-structure. The catalytic
-subunit
carries not only both substrate and allosteric sites, but also the free
radical required for catalysis, which, in class III reductases, is
located on a glycyl residue at the C terminus of the polypeptide chain.
The
-subunit generates this radical with the aid of an iron-sulfur
cluster in an anaerobic reaction that requires reduced flavodoxin and
S-adenosylmethionine (13). The glycyl radical is
oxygen-sensitive, and class III reductases can therefore operate only
in strict anaerobic bacteria or facultative aerobic bacteria growing in
the absence of oxygen.
- and small
-subunits each share
homologous amino acids. Between the classes, however, the
-subunits of the three prototypes for the classes show only very limited homology, suggesting no or only a distant evolutionary relation between
the classes. However, recent studies of class II enzymes with deep
evolutionary roots demonstrated that these enzymes and class I enzymes
share homologous amino acids for effector binding at critical positions
in the substrate specificity site (14). As for class III, excepting the
T4-induced enzyme (15), the N termini of the
-peptides containing
the hypothetical allosteric second dATP site show considerable homology
to corresponding segments of some of the new class II enzymes as well
as to that of the E. coli class I enzyme.
EXPERIMENTAL PROCEDURES
80 °C.
-D-thiogalactopyranoside was added to a final
concentration of 0.8 mM. The bacteria were incubated for another 3 h, harvested by centrifugation, and stored frozen.
Except where indicated, all further procedures were done at close to 0 °C. Extracts of the bacteria were prepared as described earlier (14) with the exception that streptomycin (at a final concentration of
1%) was included in the extraction buffer. This greatly facilitated centrifugation of the otherwise highly viscous extracts. The clear supernatant solutions were precipitated with solid ammonium sulfate to
50% saturation. After centrifugation, the precipitate was dissolved in
buffer A (30 mM Tris-HCl, pH 7.5, and 2 mm DTT) and freed
from ammonium sulfate by dialysis against the same buffer. The final solution contained ~10 mg of protein/ml. This procedure was applied to 3 g of bacteria for the purification of the L. leichmannii enzyme and to 20-50 g of bacteria for the
purification of the T. maritima and T. acidophilum enzymes.
RESULTS
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Fig. 1.
Denaturing gel electrophoresis of pure
enzymes. First lane, L. leichmannii
reductase; second lane, T. acidophilum reductase;
third lane, T. maritima reductase (TM1);
fourth lane, T. maritima reductase (TM2);
fifth lane, molecular mass markers (from top to bottom: 94, 67, 43, and 30 kDa).
Allosteric modulation of substrate specificity
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Fig. 2.
Temperature dependence of the allosteric
regulation of CDP by the T. acidophilum
reductase. The enzyme (0.4 µg/tube) was incubated with CDP
as substrate under standard conditions, except for the concentration of
ATP (closed symbols) or dATP (open symbols) given
on the separate abscissas.
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Fig. 3.
Effector binding to the L. leichmannii reductase. A, Scatchard plots of
dATP (×), dGTP ( ), and dTTP (+) binding to the enzyme (37 µg/tube). The abscissa shows the number of binding
sites/protein monomer; the ordinate shows the number of
sites divided by the concentration of the ligand. Extrapolation to the
abscissa gives the total number of sites. B, competition of
increasing concentrations of ATP (
), dGTP (
), dTTP (+), or dCTP
(
) for binding of [3H]dATP to the enzyme (41 µg/tube).
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Fig. 4.
Effector binding to the T. maritima reductase. A, Scatchard plots of
dATP (×) and dGTP ( ) binding to the enzyme monomer (1.9-3.1
µg/tube). B, competition of increasing amounts of ATP
(
), dGTP (
), dTTP (+), or dCTP (
) for binding of
[3H]dATP to the enzyme (2.8 µg/tube). C,
Scatchard plots of [3H]dATP binding to the enzyme monomer
(4.5 µg) in the presence (1.5 µM (
) and 3.0 µM
)) or absence (+) of competing dGTP.
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Fig. 5.
Effector binding to the T. acidophilum reductase. A, Scatchard plots of
dATP (×) and dGTP ( ) binding to the enzyme dimer (2.5-5.6
µg/tube). B, competition of increasing amounts of ATP
(
), dGTP (
), dTTP (+), or dCTP (
) for binding of
[3H]dATP to the enzyme dimer (3.6 µg/tube).
C, Scatchard plots of [3H]dATP binding to the
enzyme dimer (2.5-3.1 µg/tube) in the presence (1.5 µM
(
), 50 µM (
), and 200 µM (
)) or
absence (+) of competing dGTP.
DISCUSSION
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Fig. 6.
Model for effector binding to the R1 subunit
of class Ia ribonucleotide reductases. The model derives from
experiments with the E. coli protein (16). Each polypeptide
of the R1 dimer contains two separate sites with distinct functions.
The substrate specificity site binds dATP (with high
affinity), ATP, dGTP, and dTTP; the activity site binds dATP
(with lower affinity) and ATP. Effector binding to the former site
regulates the specificity of the enzyme, with dATP or ATP favoring
reduction of CDP or UDP, dGTP favoring ADP reduction, and dTTP favoring
GDP reduction. At the activity site, ATP binding increases enzyme
activity, and dATP inhibits enzyme activity.
How universal is this model? To what extent do ribonucleotide reductases belonging to other classes conform with it? For class Ia reductases, three independent lines of evidence support a common model.
Allosteric Effects on Catalysis (Criterion i)-- Where investigated, the substrate specificity of the enzymes is affected in largely the same way by a given allosteric effector. Furthermore, the activity of the enzymes is regulated positively by ATP and negatively by dATP. There are some exceptions. The Herpesvirus reductases appear to lack regulation completely (2); the phage T4 (28) and Trypanosoma brucei (29) enzymes are not inhibited by dATP.
Binding Stoichiometries between Effectors and Enzymes (Criterion ii)-- Results from limited experiments conform with the scheme of Fig. 6.
Structural Considerations (Criterion iii)-- In the structure of complexes between effectors and the R1 protein from E. coli, the activity sites were located at the far N termini, involving the 100 N-terminal amino acids (8). The specificity sites occupied a region of between 200 and 300 amino acids from the N termini and were located between the two polypeptides making up the dimer. For both sites, nucleotide-binding amino acids were identified (8). The primary structures of all class Ia reductases, except those from herpes viruses, retain these amino acids in the appropriate positions.
We now apply these three criteria to the class II reductases studied here. (i) All three regulate their substrate specificity largely according to the pattern found for class Ia reductases. The T. acidophilum reductase provides an exception in that it uses dCTP, together with dTTP, as a positive effector for the reduction of GTP. However, also the T4 enzyme, a class I reductase, employs dCTP (28). The class II enzymes are not inhibited by dATP, indicating that they lack a functional activity site. In this respect, they behave as class Ib reductases.
(ii) With respect to effector binding, the enzymes fall into two groups, with the L. leichmannii and T. maritima reductases forming one and the T. acidophilum reductase the other. The L. leichmannii and T. maritima enzymes have only one type of site that binds all nucleotides with the same stoichiometry and thus corresponds to the specificity site of class Ia and Ib reductases. The T. acidophilum reductase has two types, one capable of binding all nucleotides and the other reserved for ATP and dATP. Why does binding of dATP to this site not inhibit enzyme activity, as it does in class Ia reductases? A likely explanation is that the conformational change induced by dATP binding is not transmitted appropriately to the catalytic site. A related phenomenon is the temperature dependence of allosteric effects for the thermophilic reductases (Fig. 2) (14): at the lower temperature, the protein lacks the flexibility to induce the required allosteric transition at the catalytic site.
(iii) A comparison of the amino acid sequences of the class II reductases with that of the E. coli R1 protein shows that the T. acidophilum enzyme has many of the amino acids required for the activity site in the appropriate positions (see below), whereas the T. maritima and L. leichmannii enzymes lack this part of the sequence. This agrees with the presence of a separate dATP-binding site in the T. acidophilum enzyme, but not in the T. maritima and L. leichmannii reductases. For the specificity site, the appropriate effector-binding residues were earlier identified in the primary structures of the T. acidophilum and T. maritima enzymes, but not in the L. leichmannii reductase (14).
The evidence for an allosteric model for each of the three enzymes can be summed up as follows. The T. maritima reductase contains a specificity site according to all three criteria, but no activity site. The T. acidophilum reductase contains a specificity site according to the three criteria; it contains an activity site according to criteria ii and iii, but not criterion i, i.e. dATP is bound, but does not affect catalytic activity by the evidence we have obtained. The L. leichmannii reductase contains a specificity site according to criteria i and ii, but not criterion iii. Substrate specificity is governed by the same rules as in other reductases, but the structural basis for the specificity site is different. Note that this enzyme occupies a special position also in other respects: it is a monomer in contrast to other reductases, and it has an unusual amino acid sequence. It contains no activity site.
All three B12 reductases thus contain a specificity site obeying the rules of the model shown in Fig. 6, and their substrate specificity is regulated in the same way as that of class I and III enzymes. What differs from the model in Fig. 6 are the activity sites.
Fig. 7 shows alignments of the sequences of the 100 N-terminal amino acids of the T. acidophilum reductase with those of the corresponding sequences of the large subunits of the aerobic (class Ia) and anaerobic (class III) E. coli reductases. Both these enzymes contain a functional second dATP-binding site. The effector-binding amino acids identified by x-ray crystallography are marked in Fig. 7 (*). Identical or closely related residues occur in most of the corresponding positions of the T. acidophilum and class III reductases. Note in particular the maintenance of the motif VXKRDG (residues 5-10) with Val, Lys, and Arg contributing to effector binding. The motif is present in this position in all known class Ia and III reductases (except phage T4 (15)) and signals the occurrence of a potential second dATP site. Among the class II reductases, the motif is present also in the Pyrococcus furiosus and Mycobacterium tuberculosis enzymes, but is absent from most others.
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Leaving aside the effector-binding amino acids in Fig. 7 and looking elsewhere in the sequences, we can identify a total of 15 amino acids homologous for all three proteins. In addition, the class I and III enzymes share 7 amino acids, the class I and II enzymes share 10, and the class II and III share 19. Thus, considering only the 100 N-terminal amino acids, the T. acidophilum enzyme and the E. coli class III reductase appear to be most closely related.
We suggested earlier (3) that the three classes of ribonucleotide reductases arose during evolution from a common ancestor, most closely related to the anaerobic class III reductases, with an iron-sulfur cluster involved in free radical generation. We proposed that the appearance of molecular oxygen during evolution was the driving force for diversification. This required an enzyme capable of functioning aerobically. Class II reductases that can generate the required organic radical both anaerobically and aerobically are good candidates for the initial transition. Class I reductases probably arrived last. They require oxygen and a diferric metal center.
The present results bear on this proposed sequence of events. We
hypothesize that already an early reductase, related to present day
class III, contained two allosteric sites. One of them, the specificity
site, was maintained throughout evolution and is today found in all
reductases except in some viruses. The other, the activity site,
disappeared from some class I and II enzymes. In some cases, as in
class Ib and many class II reductases, the loss resulted from a
deletion of the N terminus. In other cases, the loss may be a result of
point mutations that disturbed the transmission of the required signal
from the allosteric to the catalytic site. An example of this is the
T. acidophilum reductase that binds dATP without giving the
appropriate catalytic response. The fact that all hitherto known class
III enzymes have maintained the structural basis for this site would be
in favor of their early existence in evolution.
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FOOTNOTES |
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* This work was supported by grants from the Karolinska Institutet, the Wallenberg Foundation, and the Wennergren Foundation (to P. R.) and by Grant PB97-0196 from the Spanish Direcciòn General de Ensenanza Superior e Investigaciòn Cientifica (to A. J.).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: Dept. of Biochemistry I, Karolinska Institute, S-17177 Stockholm, Sweden. Fax: 46 8 333525; E-mail: peter.reichard{at}mbb.ki.se.
2 A. Jordan, R. Eliasson, U. Hellman, I. Gibert, and P. Reichard, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: dNTP, deoxynucleoside triphosphate; AMPPNP, 5'-adenylylimidodiphosphate; DTT, dithiothreitol.
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REFERENCES |
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