Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.     

Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

Mouse ribonucleotide reductase control: influence of substrate binding upon interactions with allosteric effectors

Using ribonucleotide reductase encoded by vaccinia virus as a model for the mammalian enzyme, our laboratory developed an assay that allows simultaneous monitoring of the reduction of ADP, CDP, GDP, and UDP. That study found ADP reduction to be specifically inhibited by ADP itself. To learn whether this effect is significant for cellular regulation, we have analyzed recombinant mouse ribonucleotide reductase. We report that allosteric control properties originally described in single-substrate assays operate also under our four-substrate assay conditions. Three distinctions from the vaccinia enzyme were seen: 1) higher sensitivity to allosteric modifiers; 2) higher activity with UDP as substrate; and 3) significant inhibition by ADP of GDP reduction as well as that of ADP itself. Studies of the effects of ADP and other substrates upon binding of effectors indicate that binding of ribonucleoside diphosphates at the catalytic site influences dNTP binding at the specificity site. We also examined the activities of hybrid ribonucleotide reductases, composed of a mouse subunit combined with a vaccinia subunit. As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity. Surprisingly, a mouse R1/vaccinia R2 hybrid was more active than either mouse R1/R2 or vaccinia R1/R2, possibly explaining why mutations affecting vaccinia ribonucleotide reductase have only small effects upon viral DNA replication.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa. In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ. We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography. PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain. The domains are intricately linked by interdomain interactions and peptide connectors. The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members. A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site. These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site. A glutamylthioester intermediate was found in the glutaminase domain at Cys1135. The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicum.

The ribonucleotide reductase from the strictly anaerobic methanogen Methanobacterium thermoautotrophicum has been partially purified by ion-exchange and gel-filtration chromatography. Its molecular weight is estimated to be 100,000 by the latter step. Unlike all previously studied ribonucleotide reductases, the enzyme does not employ dithiol compounds such as dithiothreitol as artificial electron donors in in vitro assays. Inhibition of the enzyme by S-adenosylmethionine, oxygen, and azide further distinguishes it from the Escherichia coli anaerobic enzyme, the iron- and manganese-containing, and the adenosylcobalamin-dependent enzymes. Our preliminary results suggest that this enzyme has an activation mechanism different from the known classes of ribonucleotide reductases.     

Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.     

Di-iron-tyrosyl radical ribonucleotide reductases

New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction.     

The anaerobic ribonucleotide reductase from Lactococcus lactis. Interactions between the two proteins NrdD and NrdG.

Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.     

Structure-activity studies on synthetic peptides inhibiting herpes simplex virus ribonucleotide reductase

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) ribonucleotide reductase is formed by the association of two nonidentical subunits. A peptide corresponding to the COOH terminus of the subunit 2, Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(7-15)), has been shown to completely inhibit the reductase activity (IC50 = 36 microM) without affecting the host isoenzyme. In order to study the relationship between chemical requirements and inhibitory potencies, a series of peptides, including fragments and analogs of H2-(7-15), were synthesized. The minimum active core can be assigned to the Val-Val-Asn-Asp-Leu sequence (IC50 = 760 microM). N alpha-Extended peptides, such as Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(4-15)) and Glu-Cys-Arg-Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(1-15) ), respectively, have inhibitory potencies 2.1- and 1.4-fold greater than the nonapeptide H2-(7-15). N alpha-Deamination or acetylation of H2-(7-15) increases its potency by 1.8- and 3.0-fold, respectively, whereas amidation of the alpha-carboxylic function diminishes its activity by 3.2-fold. These results indicate that the alpha-amino group is not essential for maximum potency but suggest that a free carboxylic function is required. Substitution of Tyr7 or Ala8 by their respective D-isomer leads to a decrease of potency, suggesting that a specific conformation of the NH2-terminal portion is required to have a maximum activity. Monosubstitution in positions 11, 13, 14, and 15, by L-alanine completely abolishes activity stressing the importance of each amino acid residue contained in the minimum active core. Finally, nonapeptides corresponding to the COOH-terminal portion of the subunit 2 of Epstein-Barr and varicella-zoster virus ribonucleotide reductases also inhibit the HSV-1 reductase activity. The varicella-zoster virus nonapeptide is 4.0 times more potent than H2-(7-15), whereas the Epstein-Barr virus nonapeptide is 3.1 times less potent. These results should help us to design a new generation of potent inhibitors of herpes virus ribonucleotide reductases.     

A nitrocellulose filter binding assay for ribonucleotide reductase

Protein B1, one of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, contains two classes of binding sites for nucleoside triphosphates. One class (h-sites), with a high affinity for dATP (KD = 30 nM) regulates the substrate specificity, while the other (l-sites), with a lower affinity for dATP, regulates overall activity. These classes were defined from experiments involving equilibrium dialysis. Here we describe a sensitive alternative method to measure nucleotide binding to ribonucleotide reductases that gave the same results as equilibrium dialysis. The method involves the protein-specific binding of radioactive nucleotides to nitrocellulose filters. We believe that this method will be useful in binding studies with pure reductases from sources other than E. coli, for the characterization of mutants with changed allosteric properties, and as an assay during purification of reductases containing an h-site for dATP.     

Carbocyclic glycinamide ribonucleotide is a substrate for glycinamide ribonucleotide transformylase

The carbocyclic analog of glycinamide ribonucleotide has been synthesized from the racemic parent trihydroxy cyclopentyl amine (B.L. Kam and N.J. Oppenheimer (1981) J. Org. Chem. 46, 3268-3272). This analog was accepted as a substrate (Km = 18 microM, Vmax = 0.23 mM/min) by mammalian glycinamide ribonucleotide transformylase (EC 2.1.2.2) with an efficiency comparable to that of the natural substrate glycinamide ribonucleotide (Km = 10 microM, Vmax = 0.27 mM/min). For each molecule of 10-formyl-5,8-dideazafolate cosubstrate consumed, 0.92 molecule of N-formyl carbocyclic glycinamide ribonucleotide was produced in the enzymatic reaction, indicating a 1:1 stoichiometry. These studies afford the first alternate nucleotide substrate for glycinamide ribonucleotide transformylase and suggest that the ribose ring oxygen of glycinamide ribonucleotide is not critical for enzyme recognition and binding.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Rnr4p, a novel ribonucleotide reductase small-subunit protein.

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.     

Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC 7120 and expression in Escherichia coli

The gene for ribonucleotide reductase from Anabaena sp. strain PCC 7120 was identified and expressed in Escherichia coli. This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in E. coli, yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from E. coli extracts. Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5'-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii. In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp. strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.