Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp. Shermanii: partial purification, characteristics and role in DNA biosynthesis

Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids, P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficient P. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

Presence of oxygen-consuming ribonucleotide reductase in corrinoid-deficientPropionibacterium freudenreichii

Corrinoid-deficient Propionibacterium freuden- reichii subsp. shermanii showed adenosylcobalamin-(AdoCbl)-independent ribonucleotide reductase (RNR) activity in the presence of air. Increasing the incubation time with free access of O₂ led to an increase in RNR activity. As polarographic estimations of O₂ uptake demonstrated, AdoCbl-independent RNR activity (with ADP as substrate) in a cell-free system of corrinoid-deficient P. freudenreichii was accompanied by specific molecular oxygen consumption. The activity was not inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCCP). The activity was present in the cytoplasmic membrane-free soluble fraction of the cell extract, and it was inhibited by hydroxyurea. Manganese ions were important for the cell division of corrinoid-deficient P. freudenreichii and stimulated RNR activity after 8-hydroxyquinoline or EDTA treatment of the cell extract. We therefore concluded that P. freudenreichii is able to form DNA (deoxyribosylic precursors) using AdoCbl-dependent ribonucleotide reductase and also with an alternative AdoCbl-independent molecular-oxygen-consuming RNR system. Received: 29 May 1995 / Accepted: 14 August 1995     

Detection of a Stable Free Radical in the B2 Subunit of the Manganese Ribonucleotide Reductase (MN-RRase) of Corynebacterium ammoniagenes

Ribonucleotide reductases catalyze the irreversible reductive formation of 2′-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Coynebacterium ammoniagenes ATCC 6872. Enrichment by 2′5′-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with Superose 12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hy-droxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa 82 subunit of the Mn-RRase of Corynebacteriurn ammoniagenes.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Manganese deficiency impairs ribonucleotide reduction but not DNA replication in Arthrobacter species

Manganese deficiency induced unbalanced growth, filamentous morphology and a decrease of viability in Arthrobacter citreus ATCC 11624, A. globiformis ATCC 8010 and A. oxydans DSM 420. Under these conditions whole cells showed an inhibition of DNA formation but not of RNA synthesis. However, DNA replication still functioned when manganese-deficient cells were made permeable to and supplied with all four deoxyribonucleotides. The inhibition of DNA formation in-vivo could be traced back to impairment of DNA precursor biosynthesis as ribonucleotide reductase activity was distinctly reduced upon starvation of manganese. Both DNA formation in-vivo and ribonucleotide reductase activity were restored in the starved cultures by addition of Mn²⁺ but not of other divalent cations. In these manganese-reactivated cultures both processes were stimulated above the levels of the manganese-sufficient controls. Rifampicin or chloramphenicol (both 100 g/ml) could not suppress the rapid manganese-reactivation of cultures starved of this cation. This suggests the presence of an inactive metal-deficient ribonucleotide reductase apoenzyme in manganese-deficient cells. The presence of a manganese-dependent ribonucleotide reduction in the genus Arthrobacter besides of Brevibacterium ammoniagenes and Micrococcus luteus indicates a broad distribution of this new type of metal catalysis for DNA precursor biosynthesis in the high GC% branch of the Gram-positive bacteria.Abbreviations HU hydroxyurea - TCA trichloroacetic acid     

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)     

Ribonucleotide reductases and their occurrence in microorganisms: A link to the RNA/DNA transition

Abstract: The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors of DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.     

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.     

Impairment of DNA formation is an early event in Aspergillus niger under manganese starvation

Summary The effect of managanese on Aspergillus niger ATCC 11414 was studied during the trophophase in a citric acid-accumulating sucrosemineral medium. The absence of manganese ions restricted growth and caused characteristic morphological aberrations. Labelling with (8¹⁴-C) adenine in-vivo revealed a reversible inhibition of DNA synthesis, but not of RNA synthesis in mycelia starved of manganese. A strong inhibition of DNA synthesis in the presence of 0.2 M Mn²⁺ was observed upon addition of 200 mM hydroxyurea (HU) which is known as specific inhibitor of the ribonucleotide reductase.The hypothesis is proposed that inhibition of DNA formation in A. niger may be traced back to an impairment of manganese dependent ribonucleotide reduction which provides the monomeric precursors for DNA replication.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.     

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.     

Euglena gracilis ribonucleotide reductase: the eukaryote class II enzyme and the possible antiquity of eukaryote B12 dependence

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.     

Relationships between reversion of hydroxyurea resistance in hamster cells and the co-amplification of ribonucleotide reductase M2 component, ornithine decarboxylase and P5-8 genes

Hydroxyurea is a specific inhibitor of ribonucleotide reductase, which is a rate-limiting enzyme activity in DNA synthesis. Cells selected for resistance to hydroxyurea contain alterations in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of hamster cells has been used to isolate a stable drug resistant cell line, and two stable revertant lines with different sensitivities to hydroxyurea cytotoxicity and different ribonucleotide reductase activity levels. We show for the first time that a decrease in hydroxyurea resistance is accompanied by a parallel decline in gene copies for the M2 component of ribonucleotide reductase, ornithine decarboxylase and a gene of unknown function called p5-8, indicating that the co-amplification of the three genes is associated with drug resistance, and supporting the concept that M2, ornithine decarboxylase and p5-8 are closely linked, and form part of a single amplicon in hamster cells.     

Nitrate Reductase and Nitrous Oxide Production by Fusarium oxysporum 11dn1 Under Aerobic and Anaerobic Conditions

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively. Received: 2 February 2000 / Accepted: 28 February 2000