Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Deoxyribonucleoside-requiring mutants of Bacillus subtilis.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 degrees C but not at 25 degrees C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.     

Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibacterium ammoniagenes

The manganese-containing ribonucleotide reductase previously identified in gram-positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside-diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron-containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro. The presence of thioredoxin has been demonstrated in heat- and acid-treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12,000), isoelectric point (4.3) and enzyme-activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase-thioredoxin system in these bacteria. The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603-611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin-dependent and the iron-containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.     

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis

Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast. The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms. In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP. On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity. Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP. The relative molecular mass of the holoenzyme is about 250 000. The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy. GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonucleotide and ATP. Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents. Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (I50) is reached only at 2-6 mM hydroxyurea concentration. Up to 50% reactivation occurs spontaneously after removal of the inhibitor. In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure. Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.     

The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: Effector-induced changes

The molecular weights of Ehrlich tumor cell ribonucleotide reductase and its individual components were determined by sedimentation equilibrium in the Beckman Airfuge. The distribution of enzyme after sedimentation equilibrium was determined by measurement of the CDP reductase and ADP reductase activities associated with ribonucleotide reductase. The apparent molecular weight of the intact enzyme was 304,000 when assayed for CDP reductase and 254,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.0002. The molecular weights of the individual components of ribonucleotide reductase were determined in a similar fashion by assaying in the presence of an excess of the complementary component. The non-heme iron component had a molecular weight of 81,000 when assayed for either CDP or ADP reductase activity. The effector-binding component had an apparent molecular weight of 127,000 when assayed for CDP reductase and 95,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.004. The effectors ATP and dGTP altered the apparent molecular weights of the intact enzyme and individual components. In the presence of ATP the molecular weight of intact CDP reductase was 481,000 while the apparent molecular weight of the effector-binding component of CDP reductase alone was 418,000. In the presence of dGTP, the molecular weight of intact ADP reductase was 293,000 while the apparent molecular weight of the effector-binding component of ADP reductase alone was 154,000. These results indicate that the proportion of the non-heme iron component and the effector-binding component is not equimolar and that the composition of the enzyme is not constant but is altered by the presence of effectors. Our data also suggest that CDP reduction and ADP reduction are catalyzed by different molecular species of the enzyme which apparently have different effector-binding components.     

Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

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.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

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.     

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

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.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.     

Inhibition of iron uptake in HL60 cells by 2-formylpyridine monothiosemicarbazonato Cu(II).

The electron paramagnetic resonance (EPR) signal of the tyrosyl radical attributed to ribonucleoside diphosphate reductase decreases after treatment of promyelocytic leukemic HL60 cells with 2-formylpyridine thiosemicarbazonato copper(II) (CuL). According to EPR studies, CuL forms adducts with both histidine and cysteine-like Lewis bases associated with isolated membranes from HL60 cells. After the addition of CuL, the EPR signal for the cysteine-like adduct decreases substantially over a 15-min period. The reduction of signal is consistent with oxidation of thiols as shown by an analysis of sulfhydryl content. It is hypothesized that receptor-mediated transferrin internalization is inhibited by oxidation of critical thiols. Since the uptake of 59Fe-transferrin is greatly inhibited by the treatment of HL60 cells with CuL, the reduced uptake of iron by cells, in the presence of CuL, may lead to decreased iron availability for the activity of the M2 subunit of ribonucleotide reductase and a subsequent decrease in the tyrosyl radical signal of the enzyme. Moreover, the intact subunit M2 is no longer detected by EPR, even after the addition of excess iron.     

Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of 1-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free radical of the enzyme in an oxygen-requiring reaction

The iron chelate of 1-formylisoquinoline thiosemicarbazone is one of the most potent inhibitors known for mammalian ribonucleotide reductase. In this study, we show that the target for the drug is the tyrosine free radical of the M2 subunit of the enzyme. The radical is destroyed by the drug in a reaction which requires oxygen. After removal of the drug, the tyrosine radical and ribonucleotide reductase activity can be regenerated by incubation of the enzyme with dithiothreitol. We propose that the iron chelate of the drug binds at the active site of the enzyme, and then the ferrous form of the chelate reacts with molecular oxygen in a redox process that, via a 1-electron reduction, leads to destruction of the M2 tyrosine radical.     

The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase

An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical. The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins. After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). The nature of the third protein, provisionally named Fraction b, is unknown. The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation. Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase. Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide. Hydrogen peroxide, formed by superoxide dismutase, is also harmful. In this case, catalase present in Fraction b might protect the system. Fraction b has, however, an additional unknown function in the overall process of radical generation.     

Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.     

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.     

Mechanism of biological effect of the delta-sleep-inducing peptide includes activation of deoxyribonucleotide synthesis

To find out the mechanism of oncoprotective effects of the delta-sleep-inducing peptide, we have studied the dynamics of ribonucleotide reductase activity, which is a vital enzyme in the DNA biosynthesis, using electron paramagnetic resonance (EPR), and a concentration of low molecular mass iron complexes in the murine spleen after intraperitoneal injection of the peptide and the antitumor preparation methylnitrosourea. The data obtained give evidence on the different effects on the ribonucleotide synthesis system by the substances under study. The peptide induced significant activation of the enzyme as a consequence of activation of processes of protein synthesis. Methylnitrosourea injection leads to a decrease in the concentration of the ribonucleotide reductase active form due to destruction of the enzyme active center. The prospects of the study are discussed.     

Ribonucleotide reductase, a possible agent in deoxyribonucleotide pool asymmetries induced by hypoxia

While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.     

Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme.

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.