Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

Åberg, Anders; Hahne, Solveig; Karlsson, Matts; Larsson, Åke; Ormö, Mats; Ahgren, Agneta; Sjöberg, Britt‐Marie

doi:10.1016/0162-0134(89)84275-8

Cited by 51 publications

(92 citation statements)

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“…It contains a T7 promoter upstream of a cloning cassette (10) and has the E. coli nrdA gene, encoding protein R1, inserted (11). The plasmid pGP1-2, obtained from S. Tabor, codes for T7 RNA polymerase under the control of the P L promoter and for a heat-sensitive c1857 repressor under the control of the lac promoter (12).…”

Section: Methodsmentioning

confidence: 99%

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Birgander

Kasrayan

Sjöberg

2004

Journal of Biological Chemistry

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Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.A key enzyme of nucleic acid metabolism in the cell is ribonucleotide reductase. This enzyme, abbreviated RNR, 1 catalyzes the reaction where ribonucleotides are converted to deoxyribonucleotides, thus providing the cell with all components of DNA. RNR activity is essential for DNA synthesis and repair, but is also in need of a strict control system. An uncontrolled production of DNA precursors is devastating to the cell since the mutation frequency is increased at aberrant concentrations of dNTPs (1). In prokaryotic as well as most eukaryotic RNRs belonging to the class Ia enzymes, this is solved by an allosteric regulation controlling both the substrate specificity and the overall activity. Other classes of RNR, denoted Ib, II, and III, all perform the same reaction but operate at different conditions and via slightly different catalytic mechanisms (for a review, see Ref.2). Class Ib and II RNRs are allosterically regulated in a similar manner, and the class III enzymes are regulated approximately by the same effectors as class I RNRs (Fig. 1). In this study, all studies concern the class Ia RNR from Escherichia coli.The E. coli class Ia enzyme is composed of two dimeric proteins with different properties. The larger protein, denoted R1, contains the catalytic site and two types of allosteric sites. The catalytic mechanism involves advanced chemistry that is accomplished by a stable amino acid radical that originates from the smaller subunit, denoted R2. The fre...

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Section: Methodsmentioning

confidence: 99%

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Birgander

Kasrayan

Sjöberg

2004

Journal of Biological Chemistry

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“…Overexpression of protein was performed either in MC1009/pGP1-2 (wild-type and E238A/Y122F) or K38/pGP1-2 (E238A). The wild-type protein was grown in low iron medium containing 45.4 mM phosphate buffer, pH 7.3, 7.57 mM (NH 4 ) 2 SO 4 , 0.4% glucose, 610 M L-leucine, 406 M MgSO 4 , 50 M EDTA, 5 M CaCl 2 , 0.6 M ZnCl 2 , 60 M CuSO 4 , 0.6 M MnCl 2 , 0.75 M CoCl 2 , 5.9 M thiamine dichloride, 50 g/ml carbenicillin, and 50 g/ml kanamycin (22). The phosphate buffer, ammonium sulfate, glucose, and leucine solutions were all filtered through Chelex chelating ion exchange membranes (Bio-Rex ion exchange membrane, Bio-Rad) prior to the medium preparation.…”

Section: Methodsmentioning

confidence: 99%

Restoring Proper Radical Generation by Azide Binding to the Iron Site of the E238A Mutant R2 Protein of Ribonucleotide Reductase fromEscherichia coli

Assarsson

Andersson

Högbom

et al. 2001

Journal of Biological Chemistry

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“…2). The active site cysteines (Cys-225, Cys-439, Cys-462) (7,49,50), the proposed electron transport pathway (Tyr-730, Tyr-731) (7,10), and the two cysteines that transfer electrons between thioredoxin or glutaredoxin and the active site sulfhydryl groups (Cys-754, Cys-759) (7,49,50) are conserved in the trypanosome R1 subunit.…”

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

confidence: 99%

“…The IC 50 of the peptide was 70 M for both the mouse and the T. brucei RNR using equimolar concentrations of each subunit (0.44 M). The identical IC 50 values raised the question of whether the mouse R1 protein could form an active complex with the trypanosome R2 protein and vice versa. T. brucei has for a long time been regarded as a strictly extracellular parasite (2).…”

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

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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Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

Cited by 51 publications

References 0 publications

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Restoring Proper Radical Generation by Azide Binding to the Iron Site of the E238A Mutant R2 Protein of Ribonucleotide Reductase fromEscherichia coli

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

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