The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit

Yang, Fude; Spanevello, Rolando A.; Celiker, Incila; Hirschmann, Ralph; Rubin, Harvey; Cooperman, Barry S.

doi:10.1016/0014-5793(90)80449-s

Cited by 61 publications

(59 citation statements)

References 20 publications

(6 reference statements)

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“…The enzyme can be inhibited by nucleotide analogs, by reduction of the essential tyrosyl radical and by preventing subunit interaction [7,20,21]. In addition, interferring with the supply of reducing equivalents should strongly influence the activity of ribonucleotide reductase.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, nearly all eukaryotic proteins have a serine at this position (Ser183 in T brucei R2). Interaction between the R1 and R2 proteins is entirely accounted for by the carboxy terminus of the small subunit [15,20]. Synthetic peptides containing the seven C-terminal residues of R2 are selective inhibitors of mammalian [20], viral [21] and probably plasmodial [22] ribonucleotide reductases.…”

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

confidence: 99%

“…The heptapeptide can now be used for the preparation of an affinity matrix for purifying the R1 subunit from the parasites [20].…”

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: Discussionmentioning

confidence: 99%

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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“…Furthermore, it does not seem to be essential for catalytic activity (39). The C-terminal seven residues, which were shown to be essential for binding between the R1 and R2 proteins in the mouse RNR (35,36), show high similarity between the trypanosome and mouse R2 proteins. The only difference is that Thr-385 (mouse R2 numbering) is replaced by a serine in the T. brucei R2 protein.…”

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

confidence: 99%

“…This observation led to the develop-ment of potent peptidomimetic inhibitors of HSV RNR with antiviral activity in vivo (34). The inhibitory effect of R2 C-terminal peptides has also been demonstrated for mammalian (35,36) and E. coli class Ia (37) RNRs.…”

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confidence: 99%

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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Mono‐ and Dinuclear Palladium(II) N,S‐Heterocyclic Carbene Complexes with N Spacers and their Suzuki Coupling Activities

Yen

Koh

Huynh

et al. 2008

Chemistry An Asian Journal

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Mixed-ligand N,S-heterocyclic carbene (NSHC) complexes, trans-[PdBr(2)(NSHC)(Py)] (NSHC=3-benzyl- or 3-propyl-benzothiazolin-2-ylidene), have been obtained from bridge-cleavage reactions of the dinuclear complex, [Pd(mu-Br)Br(NSHC)](2), in pyridine at room temperature. Use of neutral N-bidentate donors (L=pyrazine, 1,2-bis(4-pyridyl)ethane, 4,4'-bipyridine and trans-1,2-bis(4-pyridyl)ethylene) yields the dinuclear spacer-bridged [Pd(2)Br(4)(NSHC)(2)(mu-L)] complexes. The X-ray single-crystal structures of the pyridyl, bridging pyrazine and 1,2-bis(4-pyridyl)ethane complexes are reported. These air-stable complexes are active in the Suzuki-Miyaura coupling reactions of selected aryl bromides. The dinuclear complexes are generally more active than their mononuclear pyridyl analogues. The benzyl derivatives consistently outperform the n-propyl counterparts.

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The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit

Cited by 61 publications

References 20 publications

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

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

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

Mono‐ and Dinuclear Palladium(II) N,S‐Heterocyclic Carbene Complexes with N Spacers and their Suzuki Coupling Activities

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