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J. Biol. Chem., Vol. 275, Issue 26, 19443-19448, June 30, 2000

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Allosteric Regulation of the Class III Anaerobic Ribonucleotide Reductase from Bacteriophage T4^*

Jessica Andersson, MariAnn Westman, Anders Hofer^§, and Britt-Marie Sjöberg^¶

From the  Department of Molecular Biology, Stockholm University, SE-10691 Stockholm, Sweden and the ^§ Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden

Received for publication, February 23, 2000, and in revised form, March 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis.

The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ribonucleotide reductase (RNR)¹ catalyzes the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides in all organisms. This is the only de novo way for the cell to make use of the deoxyribonucleotides as building blocks in DNA synthesis (1). Three classes of RNRs are known that differ in quaternary structure and cofactor requirement. Despite these differences, they all catalyze the reduction of ribonucleotides by a related radical-based mechanism (2).

All RNRs, except those of herpesviruses (3, 4), are allosterically regulated by deoxyribonucleoside triphosphates and ATP, such that DNA precursors are supplied in pools balanced according to the base composition of the different genomes (5, 6). The allosteric regulation of Escherichia coli class Ia RNR has been meticulously characterized by enzyme activity measurements, nucleotide binding, photoaffinity labeling, affinity chromatography, site-directed mutagenesis, and structure determinations (7-14). The net result of these findings is that ATP and dATP are positive effectors for reduction of pyrimidine ribonucleotides, whereas dTTP stimulates reduction of guanine ribonucleotides, and dGTP stimulates reduction of adenine ribonucleotides. Apart from this allosteric regulation, which is controlled at the so-called specificity site, class Ia RNRs contain another allosteric site denoted the overall activity site, which binds only ATP or dATP. ATP acts (directly or indirectly) as a positive effector, whereas dATP, which has a lower affinity for the overall activity site than for the specificity site, acts here as a general negative allosteric effector. The nucleotide binding component of class Ia RNRs is a homodimeric protein with four, pairwise identical, allosteric sites. The three-dimensional structure of E. coli class Ia RNR identifies two identical specificity sites located at the polypeptide interface, and two identical overall activity sites located at the N-terminal domain of each polypeptide (14).

Several RNRs conform to the allosteric substrate specificity regulation of the class Ia enzymes, whereas hitherto characterized class Ib and class II RNRs lack the overall activity control. The class Ia enzymes from bacteriophage T4 and the parasite Trypanosoma brucei also lack a general negative overall activity control (15, 16). The class III enzyme from E. coli seems most divergent, and has one allosteric site that controls reduction of pyrimidine ribonucleotides and another site that controls reduction of purine ribonucleotides (17). Recently, the allosteric regulation of the class III RNR from Lactococcus lactis was investigated and found to be similar but not identical to that of class III RNR from E. coli (18).

The three-dimensional structure of the bacteriophage T4-induced class III RNR was recently solved to high resolution (19). Interestingly, the structures of the T4 class III RNR and the class Ia RNR of the E. coli host have very similar folds and active site regions, whereas the quaternary interactions within the dimers are different (19, 20). Because one type of allosteric site is located at the dimer interface and the binding of dATP to these sites is different in the two proteins (14, 19), we were anxious to find out whether the apparent differences in mode of allosteric regulation between the well-characterized class Ia RNR and the class III enzymes were related to these structural differences.

In this study we have characterized the allosteric regulation of T4 class III RNR, currently the only class III enzyme with a known three-dimensional structure. In comparison to the amino acid sequence of other class III enzymes, and to the known three-dimensional structure of the class Ia enzyme, T4 class III RNR lacks the N-terminal domain. We show that the T4 class III enzyme has only one type of allosteric site, to which the deoxyribonucleoside triphosphates and ATP can bind. Its allosteric regulation differs from those of the E. coli and L. lactis class III enzymes and is similar to the general RNR regulation, in particular that of the class Ib and II enzymes and the class Ia RNR of T4.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- ³H-Labeled nucleotides were from Amersham Pharmacia Biotech or NEN Life Science products. They were supplied in ethanol that was removed in a SpeedVac, and the remaining nucleotide was dissolved in 25 mM unlabeled nucleotide. All unlabeled nucleotides were from Amersham Pharmacia Biotech. The filters used for the ultrafiltration assay were Ultrafree-MC filter units with polysulfone PTTK membranes from Millipore with a molecular cut-off value of 30,000.

Aerobic Overexpression and Purification of NrdD and NrdD(G580A)·NrdG-- NrdD,² or the complex NrdD(G580A)·NrdG lacking the site of the glycyl radical (21), was overexpressed and purified essentially as described previously (22, 23) with one additional purification step. The last step used an anionic exchange mono-Q column from Amersham Pharmacia Biotech, which was eluted with a gradient of NaCl and gave a sufficiently pure protein for the ultrafiltration assay.

Ultrafiltration Assay-- The ultrafiltration method for determination of nucleotide dissociation constants has been described previously (12). Initial experiments were performed in parallel with isolated NrdD wild type and with the complex NrdD(G580A)·NrdG. All binding experiments were performed aerobically, because the binding of allosteric nucleotides are not expected to involve the oxygen-sensitive glycyl radical. Similar conditions were used earlier for determining the nucleotide dissociation constants of E. coli NrdD (17). A mixture of 7-30 µM protein, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 5 mM dithiothreitol, and 1-100 µM ³H-labeled nucleotide in a final volume of 150 µl was incubated at room temperature for 5 min. An aliquot of 30 µl was withdrawn, and the rest was applied on a Millipore filter and centrifuged for 90 s at 6000 rpm. Another aliquot of 30 µl was withdrawn from the flow-through fraction, and the radioactivity of the two aliquots was quantified by liquid scintillation counting. Typically, a series of six different nucleotide concentrations were tested in each binding experiment. Binding data were evaluated either with reverse Scatchard plots (12), or by plotting [bound nucleotide]/[protein] versus [free nucleotide]. Linear regression or curve fitting to saturation curves, respectively, using Kaleidagraph software from Synergen, were used to calculate the dissociation constant (K_D) and the number of binding sites (n). Except when indicated, tabulated values are the mean of two to five independent binding experiments.

Competition Experiments-- The ultrafiltration assay was used also for competition studies. One ³H-labeled nucleotide (dATP, dGTP, or dTTP) was used at its known K_D concentration, and another unlabeled nucleotide was added at increasing concentration. The amount of bound ³H-labeled nucleotide was monitored with liquid scintillation counting. In the competition experiments with unlabeled ATP or dCTP (8 mM) the amount of Mg²⁺ used to compensate for the nucleotide quenching of magnesium ions was 20 mM.

The Binding Constant for ATP-- Ultrafiltration assays with ³H-labeled dATP, dTTP, and dGTP were performed as described earlier, and in the presence of two fixed concentrations of ATP (0.5-2 mM). This gave apparent K_D values for dATP, dGTP, and dTTP at the different ATP concentrations. The K_D, app values were plotted versus the ATP concentration according to the equation K_{D, app(dNTP)} = K_D(dNTP) (1 + [ATP]_free/K_D(ATP)), and extrapolation of the linear fits gave the K_D for ATP.

CTP Activity Assay-- The standard enzyme activity assay described in Ref. 22 was used with minor modifications. The final concentration of [³H]CTP was 1.5 mM, and the final concentration of allosteric effector was 1 mM.

Four-substrate Assay-- We used the mixed-substrate assay developed by Hendricks et al. (5), with the modifications introduced by Hofer et al. (16), and used tritium-labeled substrates for higher sensitivity. Anaerobic activity assays were as described above, but with simultaneous inclusion of [³H]CTP, [³H]ATP, [³H]GTP, and [³H]UTP in final concentrations of 1.5 mM. One unlabeled effector nucleotide (dATP, dCTP, dGTP, dTTP, or dUTP) was also added to a final concentration of 0.2 mM. Assays were stopped after different incubation times by addition of 500 µl of 1 M trichloroacetic acid. The samples were centrifuged at 14,000 rpm for 1 min at 4 °C to precipitate the proteins as pellets. The supernatants were transferred to new tubes, and the nucleotides were extracted with a freon-trioctyl mixture (1,1,2-trichlorotrifluoroethane and trioctylamine in a ratio of 3:1). The extraction was repeated (once or twice) until the pH was >5. Further pH adjustment was made by adding 1 M NH₄HCO₃, pH 8.9 (final concentration, 50 mM), and 1 M MgCl₂ (final concentration, 15 mM). The sample was then loaded onto a boronate affinity chromatography column (Affi-Gel-601 from Bio-Rad) that separates ribonucleotides from deoxyribonucleotides. The buffer was 50 mM NH₄HCO₃, pH 8.9, 15 mM MgCl₂ and the 2-ml flow-through fraction containing the deoxyribonucleotides was collected. A pH adjustment to a value between 2.5 and 3.5 was made with approximately 20 µl of 6 M HCl, and the sample was vortexed for 15 s to eliminate CO₂. A 1-ml aliquot was loaded immediately onto the high pressure liquid chromatography column (reversed phase C18 column, 4.6 × 125 mm Partisphere SAX-5 from Whatman) and eluted isocratically with phosphate buffer, pH 3.4. Identification of the nucleotide peaks was made by comparing the UV absorption profile with a standard curve, and quantification of the deoxyribonucleotide products was obtained by on-line liquid scintillation counting. Because only the substrates were tritiated and not the effectors, it was easy to distinguish between the added effector deoxyribonucleotides and the formed deoxyribonucleotide products.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding of Nucleotides to T4 NrdD-- The ultrafiltration assay by Ormö et al. (12) was used for determining the K_D value and number of binding sites for dATP, dTTP, and dGTP; a typical binding curve is shown in Fig. 1. Initially, nucleotide binding of NrdD wild type was compared with that of the NrdD(G580A)·NrdG complex. Previous studies suggest that the nucleotides bind only to the NrdD subunit (19, 23). The binding constants for NrdD wild type compared with those for the complex NrdD(G580A)·NrdG were similar for dATP, dGTP, and dTTP, respectively (Table I). The number of binding sites for dATP was higher for the complex than for the isolated NrdD, but not for dGTP or dTTP (Table I). The NrdG protein has a binding site for S-adenosylmethionine (24), and it is plausible that dATP may bind also to this site. The average K_D values for binding of dATP, dGTP, and dTTP to the isolated NrdD are summarized in Table I. The dissociation constants were all in the micromolar range, with the lowest K_D value found for dGTP, followed by dTTP, and finally dATP. The number of binding sites for these nucleotides were close to two sites per NrdD dimer (Table I).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   A typical nucleotide binding curve with dATP and NrdD wild type. Bound nucleotide/protein is plotted versus free nucleotide concentration. Vmax gives the number of binding sites per NrdD dimer and 1/2 Vmax gives the KD value. In this particular example the number of binding sites were 2.5 and the KD was 9.0 µM.

The dissociation constant for ATP was far beyond the detection limit of the ultrafiltration assay (approximately 0.1-0.2 mM)³ and had to be determined indirectly (see below). In addition, binding of dCTP and the substrate ribonucleotides, in the absence and presence of the putative prime positive effector, was far beyond our detection limits.

T4 NrdD Contains Only One Type of Allosteric Nucleotide Binding Site-- We used competition experiments to test whether dATP, dGTP, and dTTP bound to the same site in NrdD. One nucleotide, dATP, dGTP, or dTTP, was tritiated and used at a fixed concentration, and another unlabeled nucleotide was included at increasing concentrations, starting at the K_D value. The results in Fig. 2, A-C, clearly show that dATP, dGTP, and dTTP all bind to the same site in NrdD, because they can displace each other in all different combinations. The results fit very well with the individual K_D values, e.g. dGTP having the strongest binding constant is also the nucleotide most difficult to displace (Fig. 2B). We also tested ATP and dCTP as competitors to dATP, dGTP, and dTTP. As shown in Fig. 3, ATP and dCTP were able to compete with dATP but required much higher concentrations than the other nucleotides (Fig. 2, A-C). Similar results were seen in competition experiments with ATP or dCTP versus dGTP or dTTP (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Competition experiments between allosteric effector nucleotides. A, addition of tritiated dATP (8 µM) to NrdD together with increasing concentrations of unlabeled dGTP () or unlabeled dTTP (). B, addition of tritiated dGTP (0.5 µM) to NrdD together with increasing concentrations of unlabeled dATP () or unlabeled dTTP (). C, addition of tritiated dTTP (10 µM) to NrdD together with increasing concentrations of unlabeled dATP () or unlabeled dGTP (). The amount of bound tritiated nucleotide in the absence of competitor is normalized to 1 in each experiment, and the binding in the presence of the competing nucleotides is then related to this value.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Competition experiments with ATP and dCTP. Binding of tritiated dATP (8 µM) to NrdD is monitored in the presence of increasing concentrations of unlabeled dCTP () or unlabeled ATP (). The amount of bound dATP in the absence of competitor is normalized to 1, and the binding of dATP in the presence of the competing nucleotides is then related to this value.

The Binding Constant of ATP-- The competition experiments showed that ATP (and dCTP) was a competitor for the same site as dATP, dGTP, and dTTP. To determine the K_D for ATP, we used the ultrafiltration assay and measured the apparent dissociation constants for dATP, dGTP, or dTTP in the presence of several different concentrations of unlabeled ATP. The apparent K_D values were then plotted versus the concentration of ATP (Fig. 4), and from this curve it was possible to extrapolate a K_D for ATP of approximately 1.2 mM (Table I).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Indirect determination of KD for ATP. Apparent KD values, in the presence of fixed concentrations of ATP (0.5-2 mM), are plotted for dATP (), dTTP (), and dGTP (), and the KD for ATP is extrapolated from the plot as described under "Experimental Procedures."

Allosteric Regulation of CTP Reduction-- Our standard enzymatic activity assay uses CTP as a substrate. From this assay, it was clear that both ATP and dATP are positive effectors for CTP reduction (Table II). Also seen is that the enzyme has an intrinsic ability to reduce CTP in the absence of a positive allosteric effector. Addition of ATP increased the specific activity of CTP reduction 1.6 times, and addition of dATP doubled the specific activity. Addition of dTTP or dGTP resulted in a drastic decrease of the CTP reduction to 5-10% of the basal, nonregulated CTP reduction (data not shown).

Allosteric Regulation in Nucleotide Mixtures-- To identify prime allosteric effectors for the other substrates, we used the mixed-substrate assay with equal concentrations (1.5 mM) of all nucleoside triphosphates, as described previously (16). This assay gives a more "in vivo"-like situation in that reduction of all four substrates is monitored simultaneously. The conditions were the same as for the CTP activity assay, except that the effector concentrations were lower (0.2 mM) for technical reasons.

Initial experiments were designed to measure substrate reduction in the absence of effector nucleotides. In one set of experiments we included all four substrates; ATP may act both as substrate and as allosteric effector in this case. In another set of experiments we excluded ATP from the substrate mix. When only CTP, GTP, and UTP were included, only CTP was reduced (Table III). This is equivalent to the nonregulated reduction of CTP observed in the CTP activity assay (Table II). When all four substrates were included, the reduction of CTP was approximately twice as high (Table III). A corresponding increase was seen when ATP acted as a positive effector in the CTP activity assay (Table II). Likewise, CTP reduction was increased twice when dATP was used as allosteric effector in the mixed-substrate assay, but no measurable reduction of ATP, GTP, or UTP occurred (Table III).

When dGTP and dTTP were added as allosteric effectors, the reduction pattern becomes quite complex. As shown in Table III, dGTP is clearly the prime effector for ATP reduction, and dTTP is the prime effector for GTP reduction. However, other substrates are reduced as well but to a lower extent. One interpretation of the results is that dGTP and dTTP are general positive effectors for all substrates, but as can be seen in Table III, this is not the case. No GTP is reduced in the presence of dGTP, and very little CTP is reduced in the presence of dTTP. Instead, we favor the explanation that the in vivo-like conditions permit the deoxyribonucleotide products to act as positive effectors directly when they have been formed. Assays incubated for up to 20 min support this conclusion, as will be shown below. We also tested dUTP as an allosteric effector, although this nucleotide has never been observed to act as an allosteric effector for other RNRs. The results were very similar to those for dTTP, showing that the similarity of the two nucleotides transmits to their allosteric effects on T4 NrdD.

When dCTP was added as allosteric effector, both CTP and ATP were reduced (Table III). The positive effect of dCTP on ATP reduction competed with ATP to act as a positive effector for CTP reduction.

Surprisingly, we did not detect any significant UTP reduction. There is a very low level of UTP reduction at longer incubation times (0.13-0.26 nmol formed after 20 min) but not to an extent comparable to the other substrates. UTP reduction is often co-regulated with CTP reduction in other RNRs (7, 15, 17). We therefore omitted CTP from the substrate mixture, both with and without ATP or dATP as allosteric effector, but neither in the absence of nor in the presence of other effector/substrate combinations did we detect any UTP reduction (data not shown).

Time Curves Mimic an in Vivo Situation-- Further support for our interpretations of the four-substrate assays were obtained in time curve experiments with dGTP, dTTP, or dCTP as effector nucleotide. Fig. 5A shows that use of dGTP as effector initially promoted ATP reduction, whereas the resulting increase of dATP later promoted CTP reduction. After 15 min of incubation CTP reduction dominated over ATP reduction (Fig. 5A). Addition of dTTP resulted initially in GTP reduction (Fig. 5B). After 5 min of incubation, enough dGTP, which has the lowest K_D value of all allosteric effectors, had been formed to promote ATP reduction, and from 10 min onward dATP-promoted CTP reduction dominated (Fig. 5B). With dCTP as effector nucleotide, the reduction of CTP was rapid and efficient (Fig. 5C). As discussed earlier, this could represent the nonregulated CTP reduction. Formation of dATP was much slower, and even lower but significant amounts of dGTP were produced. The low reduction of ATP and GTP can probably not be ascribed to dCTP as allosteric effector, because the same pattern was seen when CTP was excluded from the substrate mix (data not shown). Fig. 5D shows the total amount of deoxyribonucleotides produced in each experiment. More NTPs were reduced with dCTP as effector than with dGTP or dTTP.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Four-substrate assays with different allosteric effectors. A, dGTP; B, dTTP; and C, dCTP as allosteric effector. The reduction of each substrate is shown as nanomoles of product made of dCTP (), dATP (), and dGTP (). ATP, which was included at a concentration of 1 mM, can act both as a substrate and as allosteric effector in these assays. D, a summary of the substrate reduction, where the summed nanomoles of dCTP, dGTP, and dTTP at each time point are shown when dGTP (×), dTTP (), or dCTP () is used as effector.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have characterized the allosteric regulation of the class III RNR from phage T4, and a summary of the results is shown in Fig. 6. The T4 class III NrdD dimer contains two equivalent allosteric nucleotide-binding sites. dATP, and high concentrations of ATP, are positive effectors for CTP reduction, dGTP (and to some extent dCTP) is a positive effector for ATP reduction, and dTTP (and dUTP) is a positive effector for GTP reduction. There is no general negative allosteric effector, and there is a substantial nonregulated reduction of CTP in the absence of allosteric effectors. The K_D values for binding of dGTP, dTTP, and dATP to T4 NrdD are in the micromolar range. This is high compared with the corresponding binding constants for class Ia RNR from bacteriophage T4 (25) and class Ia and III RNRs from the E. coli host bacterium (12, 17).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Summary of the allosteric regulation of T4 class III RNR. Black arrows denote reduction of ribonucleoside triphosphate substrates, and gray arrows denote prime positive allosteric effectors for each type of reduction. Also included is the additional metabolism of the pyrimidine deoxyribonucleotides needed to achieve dTTP and the phage-specific DNA precursor hydroxymethyl-dCTP.

The four-substrate assay (5, 16) was instrumental in demonstrating that the deoxyribonucleotide products start to compete as allosteric effectors as soon as they have formed. In this sense the four-substrate assay mimics an in vivo-like situation, and as demonstrated in this study time curves of the mixed-substrate assay are a facile way of deducing the allosteric regulatory scheme of an RNR. The results also demonstrate that the individual deoxyribonucleotide formation rates fit very well with the K_D values for the different effectors, where, e.g. dGTP, which has a much lower K_D is less efficiently formed than the other deoxyribonucleotides. The dGTP pool in uninfected E. coli is considerably lower than the other dNTP pools. Upon T4 infection, it is increased relative to the other pools, but is still the least abundant one (26). Even though the dNTP pools in general are lower during anaerobiosis than aerobiosis, in T4-infected as well as uninfected E. coli (26-28), the K_D values obtained by us (Table I) are clearly sufficient considering the in vivo concentration of each putative effector nucleotide.

From competition experiments we could conclude that ATP bound weakly (K_D 1.2 mM) to the allosteric sites in T4 NrdD. Even though its dissociation constant is orders of magnitude higher than those of the dNTPs, it may still be physiologically relevant, because the in vivo concentration of ATP is close to 3 mM (26, 29). The effect observed by high concentrations of dCTP, however, may not be physiologically relevant, because T4 genomic DNA contains hydroxymethylcytosine instead of cytosine. Consequently, T4-infected E. coli contains a pool of hydroxymethyl (hm)-dCTP instead of dCTP (30). Any formed dCTP is efficiently dephosphorylated by the T4-specific enzyme dCTPase/dUTPase to dCMP, which in turn is the substrate for the T4-specific dCMP hydroxymethylase (30). In the four-substrate assay we also observed that dUTP had the same allosteric effects as dTTP (Table III). Again, this is plausibly not physiologically significant, because both the host cell-encoded enzyme dUTPase and the T4-encoded dCTPase/dUTPase will convert any dUTP to dUMP, to enable subsequent dTTP formation via the thymidylate synthase/kinase pathway (29, 30).

Most unexpectedly, we were unable to detect any significant UTP reduction in the four-substrate assays, even when the related substrate CTP was omitted from the mixtures. In the aerobic T4 class Ia ribonucleoside diphosphate reductase, the corresponding substrate UDP has a higher K_m than the other NDP substrates, and more than two thirds of the dTTP pool is derived from CDP reduction via the dCMP deaminase and thymidylate synthase pathways (31, 32). The enzyme dCMP deaminase is allosterically regulated, positively by dCTP and hm-dCTP, and negatively by dTTP (30, 33). Likewise, the allosterically regulated enzyme dCMP hydroxymethylase is feedback-regulated by hm-dCMP (30, 33). It would thus be possible for T4 to balance the relative concentrations of dTTP and hm-dCTP via these allosterically regulated pathways, even in the absence of UTP reduction (cf. Fig. 6). The very efficient CTP reduction, compared with the reduction of GTP and ATP (Fig. 5B), speaks in favor of this suggestion. At this point we can not, however, exclude that the four-substrate assay lacks a suitable positive allosteric effector for UTP reduction, because, e.g. the allosteric effects of hm-dCTP have not been tested. However, the aerobic T4 class Ia RNR, which is very similar to the T4 class III enzyme in its allosteric behavior, responds the same to hm-dCTP as it does to the allosteric effector dCTP (15, 25).

The allosteric regulation of T4-induced class III RNR is similar to that of the class Ib RNR from Salmonella typhimurium (34) and the class II enzymes (35-38), i.e. RNRs with only one type of allosteric site (Table IV). This regulation is also seen for the class Ia RNR from phage T4 (15). E. coli NrdD (class III), on the other hand, contains two types of allosteric sites; one site regulates reduction of the pyrimidine substrates (CTP and UTP) and the other reduction of the purine substrates (ATP and GTP) (17). Class Ia enzymes also have two different types of allosteric sites; here one site regulates substrate specificity and another N-terminally located site regulates the overall activity of the enzyme by forming an active enzyme when ATP binds and an inactive enzyme when dATP binds (Table IV). Even though the allosteric regulation appears different for class Ia RNRs and E. coli class III RNR, a combination of sequence comparisons and structural considerations (14, 19) suggests that the overall location of the allosteric sites in all three RNR classes are similar. One type of allosteric site (the overall regulatory site in class Ia and the pyrimidine regulatory site in E. coli class III) is located in the N-terminal domain, and the other site (the allosteric specificity site in class Ia and the purine regulatory site in E. coli class III) is located at the dimer interface (Table IV). Class Ib enzymes and class II RNRs use only the allosteric specificity regulation at the dimer interface, and lack the overall activity control mediated by the N-terminal domain. This is achieved either by loss of physiological function of the N-terminal domain (some class II RNRs) or by loss of the N-terminal domain per se (class Ib and some class II RNRs).

The presence of only one allosteric site in T4 NrdD was expected from sequence and structure comparisons of RNRs showing that the T4 class III RNR lacks the residues corresponding to the N-terminal domain (14, 19). Also the class Ia RNR from phage T4 lacks negative allosteric control (15), even though it comprises a N-terminal domain. It has been suggested that it is advantageous for viruses to encode RNRs that lack negative allosteric control, as this will allow a high dNTP flux to multiple replication forks and promote rapid lytic growth (39). At the extreme end are the herpesviruses, whose RNRs lack both negative and positive allosteric regulation (3, 4).

Because the allosteric regulation of L. lactis was recently investigated (18), three class III RNRs can now be compared. The allosteric regulation for E. coli and L. lactis is similar though not identical, but differs from the phage RNR in that only one type of allosteric binding site is found (two are found for E. coli and L. lactis) and that dATP has a positive effect instead of being a negative inhibitor. The negative effects of dATP binding at the two different sites of the E. coli class III enzyme have not yet been experimentally distinguished from each other. Experiments where one of the sites is mutationally inactivated would be enlightening. Despite the differences between the three class III RNRs investigated so far, the common allosteric balancing of the dNTP pools by RNRs is maintained also by the class III enzymes.

	ACKNOWLEDGEMENTS

We thank Prof. I. Björk for help with derivation of equations for measuring indirect binding constants and Prof. L. Thelander for interest and support concerning the mixed-substrate assay.

	FOOTNOTES

^* This study was supported by grants from the Swedish Cancer Foundation and the Swedish Foundation for Strategic Research.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. Tel.: 46-8-164150; Fax: 46-8-152350; E-mail: bitte@molbio.su.se.

Published, JBC Papers in Press, March 23, 2000, DOI 10.1074/jbc.M001490200

² The nomenclature of the nucleotide binding components of RNRs is: class Ia, protein R1; class Ib, protein R1E; class III, NrdD. The activating component of the class III RNR is called NrdG.

³ A. Slaby and B.-M. Sjöberg, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: RNR, ribonucleotide reductase; hm, hydroxymethyl.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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