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1.
J Biol Chem ; 274(11): 7182-9, 1999 Mar 12.
Article in English | MEDLINE | ID: mdl-10066778

ABSTRACT

Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes.


Subject(s)
Ribonucleotide Reductases/metabolism , Ribonucleotides/metabolism , Vitamin B 12/metabolism , Allosteric Regulation , Amino Acid Sequence , Biopolymers , Lactobacillus/enzymology , Molecular Sequence Data , Oxidation-Reduction , Protein Binding , Ribonucleotide Reductases/chemistry , Sequence Homology, Amino Acid , Thermoplasma/enzymology , Thermotoga maritima/enzymology
2.
J Biol Chem ; 272(29): 18044-50, 1997 Jul 18.
Article in English | MEDLINE | ID: mdl-9218434

ABSTRACT

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Oxidoreductases , Proteins/metabolism , Thioredoxins/metabolism , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Glutaredoxins , Kinetics , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Proteins/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ribonucleotide Reductases/metabolism , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Thioredoxins/chemistry
3.
J Biol Chem ; 271(43): 26582-7, 1996 Oct 25.
Article in English | MEDLINE | ID: mdl-8900130

ABSTRACT

Enterobacteriaceae contain genes for three separate ribonucleotide reductases: nrdAB code for a class Ia enzyme, active during aerobiosis, nrdDG for a class III enzyme, active during anaerobiosis, and nrdEF for a cryptic class Ib enzyme. The NrdEF enzyme provides the active reductase in other, widely different bacteria. Here, we describe the allosteric regulation of the Salmonella typhimurium NrdEF enzyme. It consists of two tightly bound homodimeric proteins, R1E and R2F. Nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) regulate the substrate specificity by binding to a single site of the R1E protein (one nucleotide per polypeptide). Regulation is similar to that of the NrdAB enzyme, with one major exception: dATP stimulates reduction of CDP (and UDP) under conditions when dATP strongly inhibits all activity of the NrdAB enzyme. The nrdA-coded R1 protein contains a second binding site for dATP (and ATP) that controls general enzyme activity. All known R1E proteins lack the 50 N-terminal amino acids of R1, and we propose that the activity site is located in this area of the protein. The more sophisticated regulation of NrdAB enzymes of eukaryotes provides protection against the possibly harmful overproduction of dNTPs.


Subject(s)
Enterobacteriaceae/enzymology , Ribonucleotide Reductases/metabolism , Allosteric Regulation , Ribonucleotide Reductases/antagonists & inhibitors , Substrate Specificity
4.
J Biol Chem ; 271(15): 8779-85, 1996 Apr 12.
Article in English | MEDLINE | ID: mdl-8621514

ABSTRACT

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD- mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.


Subject(s)
Lactococcus lactis/metabolism , Oxidoreductases , Ribonucleotide Reductases/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Consensus Sequence , DNA, Bacterial/genetics , Genes, Bacterial , Glutaredoxins , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/chemistry , Proteins/chemistry , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/genetics , Sequence Alignment , Sequence Homology, Amino Acid
5.
6.
J Biol Chem ; 270(6): 2443-6, 1995 Feb 10.
Article in English | MEDLINE | ID: mdl-7852304

ABSTRACT

The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. The "activase" contains iron, required for activity. The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase. For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.


Subject(s)
Escherichia coli/enzymology , Glycine/metabolism , Ribonucleotide Reductases/metabolism , Amino Acid Sequence , Anaerobiosis , Base Sequence , DNA, Bacterial , Enzyme Activation , Free Radicals , Molecular Sequence Data , Sequence Homology, Amino Acid
7.
Proc Natl Acad Sci U S A ; 91(26): 12892-6, 1994 Dec 20.
Article in English | MEDLINE | ID: mdl-7809142

ABSTRACT

The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the R1 and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S. typhimurium nrdE and nrdF genes (R1E and R2F). They correspond to the R1 and R2 proteins. Each protein is a homodimer. Together they catalyze the reduction of CDP to dCDP, using dithiothreitol or reduced glutaredoxin, but not thioredoxin, as an electron donor. CDP reduction is strongly stimulated by low concentrations of dATP, presumably acting as an allosteric effector. Protein R2F contains an antiferromagnetically coupled dinuclear iron center and a tyrosyl free radical. The E. coli and S. typhimurium chromosome thus have maintained the information for a potentially active additional class I ribonucleotide reductase, whose role in vivo is as yet unknown. The allosteric regulation of this enzyme differs from that of the normally expressed reductase.


Subject(s)
Genes, Bacterial , Oxidoreductases , Ribonucleotide Reductases/classification , Salmonella typhimurium/enzymology , Allosteric Regulation , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Catalysis , Cytidine Diphosphate/metabolism , Electron Spin Resonance Spectroscopy , Genetic Complementation Test , Glutaredoxins , Molecular Sequence Data , Oxidation-Reduction , Proteins/pharmacology , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/metabolism , Thioredoxins/pharmacology
8.
J Biol Chem ; 269(42): 26052-7, 1994 Oct 21.
Article in English | MEDLINE | ID: mdl-7929317

ABSTRACT

The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure. Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase. The enzyme reduced ribonucleotides at a low basal rate. Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction). dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four. From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site"). This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs. The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.


Subject(s)
Escherichia coli/enzymology , Ribonucleotide Reductases/metabolism , Adenosine Triphosphate/pharmacology , Allosteric Regulation , Anaerobiosis , Binding, Competitive , Deoxyguanine Nucleotides/metabolism , Kinetics , Substrate Specificity , Thymine Nucleotides/metabolism
9.
J Biol Chem ; 269(42): 26116-20, 1994 Oct 21.
Article in English | MEDLINE | ID: mdl-7929323

ABSTRACT

The anaerobic Escherichia coli ribonucleotide reductase (class III reductase) responsible for the synthesis of the deoxyribonucleotides required for anaerobic DNA replication contains an oxygen-sensitive glycyl radical (Gly-681) suggesting involvement of radical chemistry in catalysis. The amino acid sequence of this enzyme completely differs from that of earlier described aerobic class I (prototype, aerobic E. coli) and class II (prototype, Lactobacillus leichmanii) reductases that use radical chemistry but employ other means for radical generation. Here, we study the interaction between the anaerobic E. coli reductase with the 5'-triphosphates of 2'-chloro-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, and 2'-azido-2'-deoxycytidine (N3CTP), which are mechanism-based inhibitors of class I and II reductases and, on interaction with these enzymes, decompose to base, inorganic di(tri)phosphate and 2'-methylene-3(2H)-furanone. Also, with the anaerobic E. coli reductase, the 2'-substituted nucleotides act as mechanism-based inhibitors and decompose. N3CTP scavenges the glycyl radical of the enzyme similar to the interaction of N3CDP with the tyrosyl radical of class I enzymes. However, we found no evidence for a new transient radical species as is the case with class I enzymes. Our results suggest that the chemistry at the nucleotide level for the reduction of ribose by class III enzymes is similar to the chemistry employed by class I and II enzymes.


Subject(s)
Cytidine/analogs & derivatives , Deoxycytidine/pharmacology , Escherichia coli/enzymology , Ribonucleotide Reductases/antagonists & inhibitors , Anaerobiosis , Cytidine/pharmacology , Cytosine/metabolism , Free Radicals , Structure-Activity Relationship
10.
J Bacteriol ; 175(6): 1590-5, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8449868

ABSTRACT

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dA1, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dA1 is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.


Subject(s)
Escherichia coli/enzymology , Ferredoxin-NADP Reductase/genetics , Amino Acid Sequence , Anaerobiosis , Base Sequence , Cloning, Molecular , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Escherichia coli/genetics , Ferredoxin-NADP Reductase/metabolism , Molecular Sequence Data , Oxidation-Reduction , Sequence Homology, Amino Acid , Spectrum Analysis
11.
J Biol Chem ; 267(35): 25541-7, 1992 Dec 15.
Article in English | MEDLINE | ID: mdl-1460049

ABSTRACT

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).


Subject(s)
Escherichia coli/enzymology , Ribonucleotide Reductases/metabolism , Aerobiosis , Amino Acid Sequence , Anaerobiosis , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Induction , Escherichia coli/genetics , Escherichia coli/growth & development , Kinetics , Molecular Sequence Data , Molecular Weight , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/isolation & purification , Saccharomyces cerevisiae/enzymology , Sequence Homology, Amino Acid
12.
J Biol Chem ; 267(35): 25548-52, 1992 Dec 15.
Article in English | MEDLINE | ID: mdl-1460050

ABSTRACT

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).


Subject(s)
Escherichia coli/enzymology , Ribonucleotide Reductases/metabolism , S-Adenosylmethionine/metabolism , S-Adenosylmethionine/pharmacology , Anaerobiosis , Cytidine Triphosphate/metabolism , Deoxyadenosines/metabolism , Enzyme Activation , Kinetics , Methionine/metabolism , Models, Biological , Oxidation-Reduction , S-Adenosylhomocysteine/pharmacology
13.
Exp Cell Res ; 199(1): 120-8, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1735453

ABSTRACT

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.


Subject(s)
DNA/biosynthesis , Deoxyadenine Nucleotides/metabolism , 3T3 Cells , Adenine/metabolism , Animals , Aphidicolin/pharmacology , Cell Line , Cytarabine/pharmacology , Deoxyadenine Nucleotides/biosynthesis , Deoxyadenosines/metabolism , Hypoxanthine , Hypoxanthines/metabolism , Kinetics , Mice , Pentostatin/pharmacology , Substrate Cycling , Thymine Nucleotides/metabolism
14.
Biochem Biophys Res Commun ; 180(3): 1222-6, 1991 Nov 14.
Article in English | MEDLINE | ID: mdl-1953774

ABSTRACT

Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3314-3318). Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease. The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase.


Subject(s)
Escherichia coli/enzymology , Heat-Shock Proteins , Ribonucleotide Reductases/isolation & purification , Serine Endopeptidases/isolation & purification , ATP-Dependent Proteases , Amino Acid Sequence , Anaerobiosis , Antibodies, Monoclonal , Escherichia coli/growth & development , Macromolecular Substances , Molecular Sequence Data , Sequence Homology, Nucleic Acid
15.
Eur J Biochem ; 196(1): 239-45, 1991 Feb 26.
Article in English | MEDLINE | ID: mdl-2001704

ABSTRACT

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.


Subject(s)
Deoxyribonucleotides/metabolism , Hydroxyurea/pharmacology , Animals , Cell Line , Cricetinae , DNA/biosynthesis , Deoxycytosine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Drug Resistance , Thymine Nucleotides/metabolism
16.
Proc Natl Acad Sci U S A ; 87(9): 3314-8, 1990 May.
Article in English | MEDLINE | ID: mdl-2185465

ABSTRACT

Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.


Subject(s)
Escherichia coli/enzymology , S-Adenosylmethionine/metabolism , Anaerobiosis , Chromatography, Affinity , Chromatography, Gel , Chromatography, High Pressure Liquid , Guanosine Triphosphate/metabolism , Kinetics , Peptide Fragments/isolation & purification , Peptide Mapping , Ribonucleotide Reductases/isolation & purification , Ribonucleotide Reductases/metabolism
17.
Cancer Res ; 48(13): 3681-7, 1988 Jul 01.
Article in English | MEDLINE | ID: mdl-2837322

ABSTRACT

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.


Subject(s)
DNA/biosynthesis , Deoxycytidine Kinase/deficiency , Deoxyribonucleosides/metabolism , Deoxyribonucleotides/metabolism , Phosphotransferases/deficiency , Thymidine Kinase/deficiency , Animals , Cell Line , Cricetinae
18.
Mol Cell Biol ; 7(12): 4218-24, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3437888

ABSTRACT

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.


Subject(s)
DCMP Deaminase/deficiency , Deoxyribonucleotides/metabolism , Nucleotide Deaminases/deficiency , Pyrimidines/metabolism , Animals , Cell Division/drug effects , Cell Line , Cricetinae , Cytidine Diphosphate/metabolism , DNA/biosynthesis , Deoxycytidine/metabolism , Deoxycytidine/pharmacology , Deoxycytidine Monophosphate/metabolism , Deoxycytosine Nucleotides/metabolism , Deoxyuridine/metabolism , Thymidine/metabolism , Thymidine/pharmacology , Thymidine Monophosphate/metabolism , Thymine Nucleotides/metabolism , Uridine Diphosphate/metabolism
19.
J Biol Chem ; 261(34): 16037-42, 1986 Dec 05.
Article in English | MEDLINE | ID: mdl-3536919

ABSTRACT

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.


Subject(s)
DNA/biosynthesis , Deoxyadenine Nucleotides/metabolism , Deoxycytosine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Hydroxyurea/pharmacology , Thymine Nucleotides/metabolism , Cells, Cultured , Ribonucleotide Reductases/antagonists & inhibitors
20.
Biochemistry ; 25(4): 860-7, 1986 Feb 25.
Article in English | MEDLINE | ID: mdl-3516210

ABSTRACT

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.


Subject(s)
Antibodies, Monoclonal , Escherichia coli/enzymology , Ribonucleotide Reductases/analysis , Animals , Antigen-Antibody Complex , Female , Immunoglobulin Fab Fragments , Immunoglobulin G , Kinetics , Macromolecular Substances , Mice , Mice, Inbred BALB C/immunology , Molecular Weight , Radioimmunoassay , Ribonucleotide Reductases/immunology
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