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Ribonucleoside diphosphate reductase subunits B1 and B2 and ether-permeabilized cell activities of Escherichia coli increase in parallel during thymine deprivation. Thioredoxin and thioredoxin reductase activities are not affected by thymine deprivation.
Purified calf thymus ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), showed an absolute requirement for a dithiol as hydrogen donor, whereas the natural monothiol glutathione (GSH) was inactive per se. However, a protein partially purified from thymus coupled the oxidation of GSH to the formation of deoxyribonucleotides by ribonucleotide reductase. In analogy with the ribonucleotide reductase system of Escherichia coli this protein was called glutaredoxin [Holmgren, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2275-2279]. Thymus glutaredoxin had the following properties: (i) its molecular weight determined by gel chromatography was about 12,000; (ii) it was active iwth ribonucleotide reductase in the presence of GSH, NADPH, and glutathione reductase but had no activity with NADPH and thioredoxin reductase; and (iii) it was immunologically different from thioredoxin because it did not bind to antithioredoxin immunoadsorbents. Experiments on the crossreactivity of thymus and E. coli ribonucleotide reductases and the corresponding thioredoxin and glutaredoxin systems showed essentially no specificity for the homologous thioredoxin but a high species specificity for the homologous glutaredoxin.
Ribonucleoside diphosphate reductase is an allosteric enzyme consisting of two nonidentical subunits, proteins B1 and B2. B1 contains dithiols which participate in the oxidation-reduction reactions of electron transport, while B2 contains a free radical essential for activity. Ribonucleoside diphosphates are bound to B1 but not to B2. Addition of 2'-deoxy-2'-chloro ribonucleoside diphosphates to ribonucleotide reductase irreversibly inactivates B1 without affecting B2. The reaction is specific since (a) it requires the presence of active B2, (b) it is controlled by allosteric effectors, (c) B1 is protected against inactivation by the normal substrates, and (d) the chloro-substituted nucleoside monophosphates have no effect. The inactivation of B1 is caused by a modification of the oxidation-reduction dithiols. The chloro derivatives decompose into free base, chloride ion, and 2-deoxyribose 5-diphosphate as a consequence of reaction with the enzyme. 2'-Deoxy-2"-azido ribonucleoside diphosphates cause an irreversible inactivation of B2 without affecting B1. The reaction is specific by the above criteria, indicating that the azido derivatives also bind to the active site of the enzyme. On reduction by ribonucleotide reductase, the azido derivatives function as radical scavengers and selectively destroy the free radical of B2, indicating that this radical participates in ribonucleotide reduction directly. On the basis of these results, a model of the active site of ribonucleotide reductase is proposed in which the site is formed from both B1 and B2. In the site the electron-donating oxidation-reduction active dithiols of B1 are in close contact with the free radical of B2. The azido derivative also inactivates the adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii and a ribonucleotide reductase preparation from calf thymus, indicating a general involvement of free radical intermediates in enzyme-catalyzed ribonucleotide reduction.
Each of three ribonucleoside diphosphate reductase inhibitors was used as a third drug in combination with selected antitumor platinum (Pt) agents and cyclophosphamide (CY) in the treatment of advanced L1210 leukemia in C57BL/6 x DBA/2 mice. Each was synergistic with the various Pt plus CY combinations but the effect was highly schedule dependent. The collective cure rate was 68% when hydroxyurea (HU) was given as a single injection with Pt plus CY; the cure rate was 15% when HU was administered on a divided-dose schedule with Pt plus CY. The collective cure rate was 53% when guanazole was given as a single injection with Pt plus CY, but was only 8% when it was given on a divided-dose schedule with Pt plus CY. The effect of 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, when used as a third drug with the various Pt plus CY regimens, was not schedule dependent as assessed by the collective cure rate. A therapeutic synergy between CY and each of the three ribonucleoside diphosphate reductase inhibitors was also observed.
Ribonucleoside diphosphate reductase determined by bacteriophage T4 consists of a tight complex (alpha2beta2) of the polypeptide chains alpha (Mr = 80,000 to 85,000) and beta (Mr = 35,000). The alpha2 dimer (= protein B1) was purified from Escherichia coli B infected with T4 mutant nrdB55 (Yeh, Y.C., and Tessman, I. (1972) Virology 47, 767-772) which carries an amber mutation in the gene coding for the beta polypeptide chain. Protein B1 contained binding sites for dATP, an allosteric effector of the reductase. The beta2 dimer (= protein B2) was purified by selective desorption with 1 M guanidine HCl from a dATP-Sepharose affinity column containing adsorbed native T4 ribonucleotide reductase. Protein B2, isolated this way, was enzymatically inactive due to partial loss of its iron but it could be reactivated by treatment with ferrous iron. Active protein B2 contained two atoms of non-heme iron per molecule and exhibited the optical and electron spin resonance spectra previously demonstrated in the native enzyme. The T4-induced proteins B1 and B2 were unable to reduce ribonucleotides when assayed separately but were active in combination. The proteins did not form catalytically functional hybrids with proteins B1 and B2 of Escherichia coli ribonucleotide reductase, neither did they cross-react immunologically with the latter. 5-Hydroxymethyl-dCTP, at concentrations above 10 muM, was a positive allosteric effector of T4 ribonucleotide reductase promoting the reduction of the pyrimidine ribonucleotides CDP and UDP. The nucleotide had little effect on E. coli ribonucleotide reductase.
Ribonucleoside diphosphate reductase (RDP reductase) activity was found to greatly increase after a shift to the nonpermissive temperature in Escherichia coli mutants temperature sensitive for DNA elongation (dnaE dnaG dnaZ lig) or DNA initiation (dnaA dnaC dnaI). However, the kinetics of increase in RDP reductase after a shift to nonpermissive conditions were significantly different in initiation-defective mutants compared with elongation-defective mutants. In strains without defects in DNA metabolism, the specific activity of RDP reductase was found to increase with increasing growth rate. Nutritional shifts to faster growth conditions caused cells to transiently overproduce RDP reductase before adjusting to the new steady-state conditions.
Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.
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E. coli B tsnC 7004, an E. coli B/1 mutant with normal phenotype unable to replicate phage T7 DNA [Chamberlin, M. (1974)J. Virol. 14,509-516], contained no detectable level of thioredoxin when assayed with ribonucleotide reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Gently lysed E. coli tsnC 7004 cell extracts reduced CDP when supplemented with NADPH as efficiently as the parent strain E. coli B/1 despite the lack of thioredoxin, indicating the presence of another hydrogen transport system. This could be divided into two parts by heat treatment at 85degrees; one heat-stable fraction, which was active in the presence of dithiothreitol or glutathione, and one heat-labile fraction. Addition of yeast glutathione reductase [NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2] to the heated extracts restored full activity. The results demonstrate a novel hydrogen transport system in E. coli consisting of NADPH, glutathione, glutathione reductase, and a heat-stable enzyme called "glutaredoxin". Reduced glutathione at physiological concentrations functions as hydrogen donor for ribonucleotide reduction only in the presence of glutaredoxin. Glutaredoxin was not reduced by E. coli thioredoxin reductase (NADPH:oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) and showed no crossreaction with antibodies against thioredoxin. These results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis.
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Ribonucleoside diphosphate reductase from Escherichia coli consists of a 1/1 complex of two nonidentical subunits called proteins B1 and B2. The enzyme reduces the four common ribonucleoside diphosphates to the corresponding deoxyribonucleotides and is allosterically regulated by nucleoside triphosphates which influence its substrate specificity as well as its overall activity. The B1 subunit contains binding sites for the effectors while B2 contains iron and an organic free radical essential for catalytic activity. We now establish that only protein B1 binds substrates. Competition experiments support the presence of two identical substrate binding sites, distinct from the effector binding sites. The catalytic site of the enzyme thus is formed from both the B1 and B2 subunits. Dissociation constants for substrates ranged from 2 X 10(-5) to about 10(-3) M. In all cases effectors decreased these constants in agreement with their influence on the substrate specificity of ribonucleotide reductase, but did not induce cooperative effects. The increase in binding was pronounced at 20 degrees but only marginal at 0 degrees. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed sharp breaks at 12 degrees. The temperature effects can be interpreted as a conformational change occurring in the structure of protein B1 at the critical temperature.
The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, we have selected, cloned, and characterized a mutant mouse T cell lymphoma (S49) completely deficient in PNPase. Of the four substrates of PNPase, only deoxyguanosine at low concentrations is toxic to the PNPase-deficient (NSU-1) cells. In order to delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to deoxyguanosine, we have isolated a series of secondary mutants resistant to deoxyguanosine from the PNPase-deficient line. One of these mutants is defective in its ability to transport deoxyguanosine into the cell. A second type of mutant cannot phosphorylate the deoxyguanosine and is totally deficient in deoxycytidine kinase activity. A third type of mutant (NSU-1-dGuo-L) can both transport and phosphorylate deoxyguanosine and accumulates dGTP. However, unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous deoxyguanosine. This observation is accounted for by the fact that the reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP.Thus, in order to exert its toxicity deoxyguanosine must be transported into the cell, be phosphorylated by deoxycytidine kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.
In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.
HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.
Reduced thioredoxins from microbial and plant cells, both of cytoplasmic or chloroplast origin, are interchangeable in stimulating such diverse enzyme activities as ribonucleoside diphosphate reductase (E. coli), PAPS sulfotransferase (Synechococcus), and fructose-1,6-bis-phosphatase (from spinach) in vitro. It is suggested that reduced thioredoxins are unspecific, multifunctional cellular proteins while in contrast the oxidized froms require specific enzymes for their reduction.