Mouse ribonucleotide reductase

Rova, U., Goodtzova, K., Ingemarson, R., Behravan, G., Graslund, A., and Thelander, L., 1995, Evidence by site-directed mutagenesis supports long-range electron-transfer in mouse ribonucleotide reductase Biochemistry 34 4267n4275. 

Davydov, A., Schmidt, P. P., and Gr%oslund, A., 1996a, Reversible red-ox reactions of die diiron site in the mouse ribonucleotide reductase R2 protein. Biochem. Biophys. Res. Comimm. 219 213n218. 

Nyholm, S., Thelander, L., and Gr%oslund, A., 1993, Reduction and loss of the iron center in the reaction of the small subunit of mouse ribonucleotide reductase with hydroxyurea. Biochemistry 32 11569911574. 

Ochiai, E., Mann, G. J., Gr%oslund, A., and Thelander, L., 1990, Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase. J. Biol. Chem. 265 15758915761. 

Figure 4 a) X-band EPR spectra of tyrosyl free radical in (i) E. coli, (ii) Mycobacterium tuberculosis, and (iii) mouse ribonucleotide reductase R2 proteins (1 7). All spectra were obtained under nonsaturation conditions at 20 K. b) Spin density distribution of the unpaired electron obtained from Isotope-labeling EPR studies, c) The distances between the phenolic oxygen of tyrosyl radical and the nearest Fe ion deduced from the relaxation properties of the tyrosyl radicals. 

Filatov D, Ingemarson R, Johansson E, Rova U, Thelander L. 1995. Mouse ribonucleotide reductase from genes to proteins. Biochem Soc Trans 23 903-905. 

G, Graslund A. 1999. The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase EPR and electron nuclear double resonance studies on a new transient tryptophan radical. J Biol Chem 274 17696-17704. 

Nielsen BB, Kauppi B, Thelander M, Thelander L, Larsen IK, Eklund H. 1995. Crystallization and crystallographic investigations of the small subunit of mouse ribonucleotide reductase. FEBSLett 373 310-312. 

Bighno D, Schmidt PP, Reijerse EJ, Luhitz W. 2006. PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase. Phys Chem Chem Phys 8 58-62. 

Jenh C-H, Geyer PK, Johnson LF (1985) Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse line. Mol Cell Biol 5 2527-2532 Jong AY, Yu K, Zhou B, Frgala T, Reynolds CP, Yen Y (1998) A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5 62-68... 

Specific inhibitors of ribonucleotide reductase such as hydroxyurea (type 1 in the list) and azidocytidine (type 3) have already been used in cancer therapy. Hydroxyurea sensitivity is so strongly a characteristic of the class IRNR enzymes that it is commonly used as a diagnostic for this class 105). The functional tyrosyl radical is effectively reduced by hydroxyurea 106-108). In mouse and HSVl enzymes, the iron center is also reduced and labilized in this process 109), whereas in... 

EPR study of the mixed-valent diiron sites in mouse and herpes simplex virus ribonucleotide reductases. Effect of the tyrosyl radical on structure and reactivity of the diferric center. Biochemistry 36, 9093-9100. 

Fig. 3a-c. ESR spectra of the tyrosine free radicals in ribonucleotide reductases, a protein subunit B 2 of . coli enzyme, measured at 86 K "> b bacteriophage T4 enzyme, at 77 K c hydroxyurea-resistant, reductase-overproducing mouse 3T6 cells, at 32 K ... 

The topology of the radical site pocket of calf thymus and mouse fibroblast ribonucleotide reductase was recently probed with a series of hydroxamate inhibitors of increasing bulkiness and will be discussed in the following section. Other mammalian sources from which ribonucleotide reductases have been isolated and more or less purified include rat Novikoff hepatoma and regenerating rat liver" rabbit bone mar-row 5,66) Ejjj-iich ascites tumor cells of mice and cultured human lymphoblast cells Some of their properties are described in Table 2. Many more animal and human cells were assayed for enzyme activity, frequently in mutant cell lines, to test for cell cycle dependence, mechanisms of metabolic regulation, drug resistance, and correlation with tumor growth rates. Representative studies of this kind, which rapidly expand in number, are summarized in Table 3. 

The activation and inhibition patterns established for calf thymus and human ribonucleotide reductases resemble that of the E. coli enzyme in many ways (Table 6). Two different classes of effector sites are not only likely from the analogous stimulatory and inhibitory effects of deoxyribonucleotides and ATP on substrate reduction. In certain mouse cell lines two regulatory protein domains on reductase subunit M1 have indeed been identified which are mutated independent of each other On the other hand phage T4 enzyme, with closely comparable protein structure, behaves different in that the inhibitory effect of dATP is missing, indicating alteration of the activity sites. 

The idea of common ancestry of the different ribonucleotide reductases is difficult to test at present because protein sequencing studies have not yet begun. Exchangeability of subunits, possible among the calf thymus and mouse enzymes , has not much been tried. We have seen small but significant stimulation of enzyme activity when the separated, inactive subunits U1 of algal (Scenedesmus) ribonucleotide reductase and B 2 of E. coli were recombined, but not in the reverse combination (B1 -t- U2) . The many individual differences in enzyme structure like Mg or Ca " requirement for subunit interaction, variations in the radical environment as expressed in slightly different ESR spectra (Fig. 3), or details of allosteric effector pattern, do not in principle contradict our reductase model but will in reality severely limit its experimental verification. 

The effects of hydroxyurea in the purified ribonucleotide reductase systems of E. coU, phage T4, calf thymus and mouse cells have been described above (p. 36, 42). Inhibition of substrate reduction in vitro (I50 = 2 - 3 10 ) is accompanied by loss of the tyrosyl radical, but not iron from E. coli subunit B2. Studies with substituted hydroxyl-amines and hydroxamates showed good correlation between their ability to undergo one-electron oxidation and enzyme inhibition, unless branched substituents prevented interaction with the protein (Table 8) Thus the mode of inhibition of E. coli ribonucleotide reductase is essentially solved Within steric restrictions of accessibility to the active site the compounds donate an electron to the enzyme s free radical, producing an inactive protein with still intact binuclear iron complex (Eq. VI). This process is irreversible in vitro until iron is removed, and then reintroduced with Fe(II)ascorbate in the presence of oxygen, whereupon radical and enzyme activity reappear. No other enzyme of E. coli has been found to be inactivated by hydroxyurea. 

The in vitro reduction of CDP, ADP, and GDP by different mammalian enzymes is as sensitive to hydroxyurea (I50 = 2 - 4 x 10" M) as with E. coli enzyme . The tyrosine radical of mouse fibroblast ribonucleotide reductase disappears upon addi-... 

In HeLa ceils hydroxyurea is an efficient inhibitor of histone synthesis. This action requires protein synthesis and leads to rapid disappearance of cytoplasmatic histone mRNA The effect is not specific for hydroxyurea since suppression of DNA synthesis by arabino-cytosine or temperature-sensitive mutations yields analogous results. Similarly, the synthesis of some enzymes necessary for DNA replication and active in S-phase is altered by hydroxyurea. Increased activity of ribonucleotide reductase in HeLa and in hamster cells and of the salvage enzyme thymidine kinase in HeLa cells and KB cells has been observed, probably as a consequence of the increased fraction of cells in S-phase. Repression occurs for thymidine kinase in human lymphocytes and for ornithine decarboxylase in Chinese hamster fibroblasts whereas no or only slight effects were seen on ribonucleotide reductase in hamster fibroblasts , on thymidylate synthase in extracts from synchronous mouse cells " , and on DNA polymerase in rabbit kidney cells or HeLa cells . ... 

Fig. 2.29 Four-pulse X-band PELDOR of mouse R2 ribonucleotide reductase protein experimental blue line) and simulated red line) spectrum. The modulations are due to the interaction between two tyrosyl radicals at a distance of 3.25 0.05 nm. The figure is reproduced from Ref [58] with permission from the Royal Society of Chemistry...

Ribonucleotide reductase activity has been related to proliferation of animal cells in several ways. Turner et al. (34) found that the ribonucleotide reductase activity present in cultured mouse fibroblasts (L cells) was related to the mitotic cycle, being detectable only in the phase of DNA synthesis. The reductase is scarcely detectable in adult liver however, it is present in tumors and in proliferating tissues and, as Elford et al. 16), have reported, an excellent correlation exists between reductase activity and growth rate in a series of rat hepatomas. Evidently, the activity of ribonucleotide reductase varies in response to the cellular demand for DNA materiel. 

It has been reported recently that mycophenolic acid inhibits DNA synthesis by fibroblasts. This is due to an effect of the antibiotic on an early stage of the biosynthesis of purine nucleotides blocking the enzyme IMP-NAD oxidoreductase . Toyocamycin and tubercidin were found to cause accumulation in mammalian cells of the 45 S RNA precursor of the 28 S and 18 S ribosomal RNA. The antibiotic sangivamycin was shown to be phosphorylated by enzyme extracts from mouse liver. Sangivamycin triphosphate was shown to be a substrate for ribonucleotide reductase but not for deamination . ... 

Graslund A. 2002. Ribonucleotide reductase Kinetic methods for demonstrating radical transfer pathway in protein R2 of mouse enzyme in generation of tyrosyl free radical. In Enzyme kinetics and mechanism, Pt F detection and characterization of enzyme reaction intermediates, pp. 399 14. New York Academic Press. 

Thelander M, Graslund A, Thelander L. 1985. Subunit M2 of mammalian ribonucleotide reductase characterization of a homogeneous protein isolated from M2-oveiproducing mouse ceMs.J Biol Chem 260 2737—2741. 

Strand KR, Karlsen S, Andersson KK. 2002. Cobalt snbstitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state spectroscopic and structural evidence for a ferromagneticaUy conpled dinnclear cobalt cluster. J Biol Chem 277 34229-34238. 

Atta M, Andersson KK, Ingemarson R, The lander L, Graslund A. 1994. EPR Studies of mixed-valent [Fe Fe ] clusters formed in the R2 subunit of ribonucleotide reductase from mouse or herpes simplex virus mild chemical reduction of the diferric centers. J Am Chem Soc 116 6429-6430. 

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