The Three-dimensional Structure of Mammalian Ribonucleotide Reductase Protein R2 Reveals a More-accessible Iron-radical Site than R2

Kauppi, Björn; Nielsen, Bettina Bryde; Ramaswamy, S.; Larsen, Inger Kristin; Thelander, M; Thelander, Lars; Eklund, Hans

doi:10.1006/jmbi.1996.0546

Cited by 138 publications

(152 citation statements)

References 39 publications

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“…The conclusion is in accordance with the sensitivity of trypanosomes towards hydroxyurea [17][18][19] although at low concentrations of hydroxyurea inhibition of ribonucleotide reductase seems not to be the primary effect [19]. The sensitivity of ribonucleotide reductases from different species towards radical scavengers varies remarkably and may be due to the accessibility of the iron/radical center [14]. In the E. coli enzyme a channel which connects the surface with the proposed oxygen reaction site is shielded by a tyrosine residue.…”

Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinssupporting

confidence: 74%

“…Other residues in the vicinity of the tyrosine are not strictly conserved. As outlined by Kauppi et al [14] the tyrosine environment in mouse R2 is more hydrophobic than that in the E. coli enzyme since two Phe and a Met replace a Tyr, Gln ( Contmue~ and Set. In the 72 brucei sequence, Phe80, Phe81 and Thr122 occupy the respective positions.…”

Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinsmentioning

confidence: 98%

“…1). Asp85, Glu116, His119, Glu178, Glu212 and His215 correspond to the iron ligands in mouse and E. coli R2 [7,13,14].…”

Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinsmentioning

confidence: 99%

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Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

et al. 1997

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As a first step towards the understanding of the trypanosomal synthesis of deoxyribonucleotides we describe the cloning of the gene of ribonucleotide reductase subunit R2 from 72 brucei hrucei and its overexpression in E. coli. Materials and methods DNA and RNA preparation2;4 l08 long slender 72 brucei cells were suspended in 10 mM Tris-HC1, 250 mM NaCI, 0.5% Nonidet P-40, pH 8.0. After 5 rain incubation on ice the suspension was centrifuged and the cell pellet was homogenized in 200 lal 10 mM Tris-HC1, 10 mM NaC1, 10 mM EDTA, 0.5% SDS, pH 8.0. 50 lag proteinase K were added and the mixture was incubated for 1 h at 37°C. DNA was purified by phenol extraction.RNA was isolated from bloodstream long slender and short stumpy as well as procyclic stages of 72 brucei using the RNA Easy Kit (Qiagen) according to the instructions of the manufacturer.

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Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinssupporting

confidence: 74%

Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinsmentioning

confidence: 98%

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Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

et al. 1997

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“…The N terminus of the mouse R2 protein up to amino acid residue 65 is disordered and not visible in the crystal structure (28). Furthermore, in NMR studies, a number of resolved resonances not belonging to the highly mobile C terminus may be due to f lexible N-terminal residues (29).…”

Section: Discussionmentioning

confidence: 99%

Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis

Chabes

Pfleger

Kirschner

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosis͞G 1 in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G1, may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G 0͞G1.

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“…A striking difference between the mouse and the T. brucei R2 proteins is that the mouse R2 protein is larger due to 53 extra residues in the N terminus. This is probably a highly flexible part of the protein, since it cannot be detected in the crystal structure (47). Furthermore, it does not seem to be essential for catalytic activity (39).…”

Section: Cloning and Sequencing Of T Brucei R1 And R2 Cdnasmentioning

confidence: 98%

Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

Hofer

Schmidt

Gräslund

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the de novo synthesis of deoxyribonucleotides by directly reducing ribonucleotides to the corresponding deoxyribonucleotides. To keep balanced pools of deoxyribonucleotides, all nonviral RNRs studied so far are allosterically regulated. Most eukaryotes contain a class I RNR, which is a heterodimer of two nonidentical subunits called proteins R1 and R2. We have isolated cDNAs encoding the R1 and R2 proteins from Trypanosoma brucei. The amino acid sequence identities with the mouse R1 and R2 subunits are 58% and 63%, respectively. Recombinant active trypanosome R1 and R2 proteins were expressed in Escherichia coli and purified. The R2 protein contains an iron-tyrosyl free radical center verified by EPR spectroscopy and iron analyses. Measurement of cytidine 5 -diphosphate reduction by the trypanosome RNR in the presence of various allosteric effectors showed that the activity is highest with dTTP, dGTP, or dATP and considerably lower with ATP. The effect of dGTP is either activating (alone) or inhibitory (in the presence of ATP). Filter binding studies indicated that there are two classes of allosteric effector binding sites that bind ATP or dATP (low-affinity dATP site) and ATP, dATP, dGTP, or dTTP (high-affinity dATP site), respectively. Therefore, the structural organization of the allosteric sites is very similar to the mammalian RNRs, whereas the allosteric regulation of cytidine 5 -diphosphate reduction is unique. Hopefully, this difference can be used to target the trypanosome RNR for therapeutic purposes.Sleeping sickness is a major health problem in a predominant part of sub-Saharan Africa. The disease, caused by a unicellular eukaryote called Trypanosoma brucei (1), is usually fatal if left untreated, and Ϸ25,000 new cases are diagnosed each year. However, since only a small fraction of the population in high risk areas is under surveillance, the World Health Organization has estimated the real figure to be Ϸ300,000. ‡ The current chemotherapy of sleeping sickness suffers from various limitations. Many of the compounds used are highly toxic and there is also a widespread drug resistance among the trypanosomes (2, 3).Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. There are three different classes of RNRs (4). The substrate, which is a nucleoside diphosphate for the class I enzymes, is reduced through a radical mechanism. Class I RNR is a heterodimeric enzyme of ␣ 2 ␤ 2 type. The large R1 subunit binds substrates and allosteric effectors, while the small R2 subunit contains a tyrosyl radical (5), generated by a binuclear ferric iron center. The tyrosyl radical in the R2 protein is linked to the active site in the R1 subunit through a hydrogen-bonded long-range electron transport chain (6-10). Class II RNR generates a radical from 5Ј-deoxyadenosylcobalamin (11, 12) and class III RNR, which only works under anaerobic conditions, contains a stable glycyl radical (13). Class I and to s...

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The Three-dimensional Structure of Mammalian Ribonucleotide Reductase Protein R2 Reveals a More-accessible Iron-radical Site than R2

Cited by 138 publications

References 39 publications

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis

Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

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