Comprehensive Model for Allosteric Regulation of Mammalian Ribonucleotide Reductase:  Refinements and Consequences

Kashlan, Ossama B.; Cooperman, Barry S.

doi:10.1021/bi020634d

Cited by 84 publications

(144 citation statements)

References 34 publications

(65 reference statements)

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“…Here the mutation at the allosteric activity site led to a decreased binding of dATP also at the specificity site (25). However, in a recent study on the mouse enzyme with the D57N mutation, Kashlan and Cooperman (26) did not see any effect on binding to the specificity site. The mouse and the E. coli enzymes are not necessarily regulated in an identical manner.…”

Section: Discussionmentioning

confidence: 83%

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: Discussionmentioning

confidence: 83%

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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“…R1 is composed of two 85-kDa monomers and R2 is composed of two 43.5-kDa monomers (Figure 2). The active complex of the mouse RNR has been reported to have an a2b2 and a6b6 composition (Scott et al, 2001;Kashlan and Cooperman, 2003). Structures at atomic resolution of E. coli R1 (Uhlin and Eklund, 1994), and mouse (Nielsen et al, 1995;Strand et al, 2004), and yeast R2s Sommerhalter et al, 2004) have been published.…”

Section: Studies On the Mechanism Of Radical Initiation In R1mentioning

confidence: 99%

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Bennati

Lendzian²,

Schmittel³

et al. 2005

Biological Chemistry

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Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid-or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes.

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“…The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42).…”

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confidence: 99%

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

Huang

2008

Molecular and Cellular Biology

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Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase (RNR). RNR is controlled via transcription, protein inhibitor association, and subcellular localization of its two subunits, R1 and R2. Saccharomyces cerevisiae Sml1 binds R1 and inhibits its activity, while Schizosaccharomyces pombe Spd1 impedes RNR holoenzyme formation by sequestering R2 in the nucleus away from the cytoplasmic R1. Here we report the identification and characterization of S. cerevisiae Dif1, a regulator of R2 nuclear localization and member of a new family of proteins sharing separate homologous domains with Spd1 and Sml1. Dif1 is localized in the cytoplasm and acts in a pathway different from the nuclear R2-anchoring protein Wtm1. Like Sml1 and Spd1, Dif1 is phosphorylated and degraded in cells encountering DNA damage, thereby relieving inhibition of RNR. A shared domain between Sml1 and Dif1 controls checkpoint kinase-mediated phosphorylation and degradation of the two proteins. Abolishing Dif1 phosphorylation stabilizes the protein and delays damage-induced nucleus-to-cytoplasm redistribution of R2. This study suggests that Dif1 is required for nuclear import of the R2 subunit and plays an essential role in regulating the dynamic RNR subcellular localization.Maintenance of genomic stability depends on faithful replication of DNA and repair of lesions after damage. Fidelity of both DNA replication and repair is influenced by perturbation in the sizes and relative ratios of cellular deoxynucleotide triphosphate (dNTP) pools. Ribonucleotide reductase (RNR) catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms and is largely responsible for maintaining cellular dNTP pools (34). As maximal DNA synthesis requires high concentrations of dNTPs, the RNR activity plays an important role in cell proliferation (31). On the other hand, increased RNR activity has been associated with malignant transformation and resistance to chemotherapy (12,14,25,56).The class I RNR holoenzymes are commonly found in eukaryotes and eubacteria and comprise two subunits, R1 and R2 (34). The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42). Mammalian genomes contain a single R1 gene and two R2 genes; the cell cycle-regulated RRM2 is responsible for providing dNTPs in actively dividing cells, and the DNA damage-inducible p53R2 is required for replenishing dNTP pools in cells under genotoxic stress (9,22,44). Loss of p53R2 causes mitochondrial DNA depletion and increased apoptosis (22). The budding yeast Saccharomyces cerevisiae has two R1 genes, RNR1 and RNR3. RNR1 is essential for mitotic growth, while RNR3 is high...

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Comprehensive Model for Allosteric Regulation of Mammalian Ribonucleotide Reductase: Refinements and Consequences

Cited by 84 publications

References 34 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

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

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