Ribonucleotide reductase (RNR), also known as ribonucleoside diphosphate reductase, is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. It catalyzes this formation by removing the 2'-hydroxyl group of the ribose ring of nucleoside diphosphates (or triphosphates depending on the class of RNR). This reduction produces deoxyribonucleotides. Deoxyribonucleotides in turn are used in the synthesis of DNA. The reaction catalyzed by RNR is strictly conserved in all living organisms. Furthermore, RNR plays a critical role in regulating the total rate of DNA synthesis so that DNA to cell mass is maintained at a constant ratio during cell division and DNA repair. A somewhat unusual feature of the RNR enzyme is that it catalyzes a reaction that proceeds via a free radical mechanism of action. The substrates for RNR are ADP, GDP, CDP and UDP. dTDP (deoxythymidine diphosphate) is synthesized by another enzyme (thymidylate kinase) from dTMP (deoxythymidine monophosphate).

Structure

Ribonucleotide reductases are divided into three classes. Class I RNR enzymes are constructed from large alpha subunit and small beta subunits which associate to form an active heterodimeric tetramer. By reducing NDPs to 2'-dNDPs, the enzyme catalyses the de novo synthesis of deoxyribonucleotides (dNTPs), which are precursors to DNA synthesis and essential for cell proliferation. Class II RNRs produce a 5'-deoxyadenosyl radical by homolytic cleavage of the C-Co bond in adenosylcobalamin. In addition, Class III RNRs contain a stable glycyl radical.

Humans carry Class I RNRs. The alpha subunit is encoded by the RRM1 gene while there are two isoforms of the beta subunit, encoded by the RRM2 and RRM2B genes:

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Each Class I alpha monomer consists of three domains:

  • one mainly helical domain comprising the 220 N-terminal residues,
  • a second large ten-stranded α/β structure comprising 480 residues,
  • and a third small five-stranded α/β structure comprising 70 residues.

In Pfam, the second domain has been interpreted as two separate domains:

  • a shorter all-alpha N-terminal domain,
  • and a longer barrel C-terminal domain.

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The Class I beta subunit usually contains a di-metal center and a stable tyrosyl radical. In humans, the beta subunit relies on a di-iron cofactor. In E. coli, the tyrosyl radical is located at position 122 (Y122) providing the stable radical for the Class I RNR2 subunits. In A. aegypti, this tyrosyl radical is located at position 184 (Y184). The tyrosyl radical is deeply buried inside the protein in a hydrophobic environment, located close to the iron center that is used in the stabilization of a tyrosyl radical. The structure of two μ-oxo-linked irons is dominated by ligands that serve as iron binding sites: four carboxylates <nowiki>[</nowiki>aspartate (D146), glutamate (E177, E240, and E274)<nowiki>]</nowiki> and two histidines (H180 and H277). It is a paralog of the yeast RNR1, which likely arose from a whole genome duplication event. The RNR complex catalyzes the rate-limiting step in dNTP synthesis, regulated by DNA replication and DNA damage checkpoint pathways via localization of small subunits.

Function

thumb|left|400px|Mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. (adapted from Nelson & Cox, 2000). (1) an electron transfer on the RNR2 subunit activates a RNR1 cysteine residue in the active site with a free radical; (2) the free radical forms a stable radical on C-3, and cysteine radical removes a hydrogen atom; (3) cation is formed on C-2 by transferring a hydrogen from a dithiol group and is stabilized by the radical, resulting in the loss of H2O from C-2; (4) a hydrogen is transferred from the dithiol group to reduce the cation C-2; (5) the C-3 radical is reduced by the hydrogen removed in step 2, and the tyrosyl radical is generated; (6) redoxins transfer two hydrogen to the [[disulfide group that restores the original configuration.]]

The enzyme ribonucleotide reductase (RNR) catalyzes the synthesis of dNDPs. A free radical abstracts a hydrogen from a C-H bond of the ribose. Following a single reduction, RNR requires electrons donated from the dithiol groups of the protein thioredoxin or thioredoxin like proteins eg NrdH. Regeneration of thioredoxin occurs when nicotinamide adenine dinucleotide phosphate (NADPH) provides two hydrogen atoms that are used to reduce the disulfide groups of thioredoxin. interacting with enzyme mutants. The next step is the oxidation of the anionic disulfide bridge, with concomitant reduction of the substrate, generating 5. The spin density shifts from the sulphur atoms to the C-3' atom of the substrate, with simultaneous proton transfer from Glu441 to carbon C-3'. The last step is the reverse of the first step and involves a hydrogen transfer from Cys439 to C-3', regenerating the initial radical and resulting in the final product 6.

Theoretical models of some steps of these mechanisms using the full model of the R1 protein can be found at the studies performed by Cerqueira et al..

Regulation

thumb|left|400px|Regulation of class I RNR. Class I RNRs are activated by binding ATP or inactivated by binding dATP to the activity site located on the RNR1 subunit. When the enzyme is activated, substrates are reduced if the corresponding effectors bind to the allosteric substrate specificity site. A = when dATP or ATP is bound at the allosteric site, the enzyme accepts UDP and CDP into the catalytic site; B = when dGTP is bound, ADP enters the catalytic site; C = when dTTP is bound, GDP enters the catalytic site. The substrates (ribonucleotides UDP, CDP, ADP, and GDP) are converted to dNTPs by a mechanism involving the generation of a free radical.

Class I RNR comprises RNR1 and RNR2 subunits, which can associate to form a heterodimeric tetramer. Additionally, it is important that the activity of ribonucleotide reductase be under transcriptional and post-transcriptional control because the synthesis of damage-free DNA relies on a balanced pool of deoxyribonucleotides. Eukaryotic cells with class IA reductases have a mechanism of negative control to turn off synthesis of dNTPs as they accumulate. This mechanism protects the cell from toxic and mutagenic effects that can arise from the overproduction of dNTPs because changes in balanced dNTP pools lead to DNA damage and cell death. Although, the overproduction of dNTPs or an unbalanced supply of them can lead to misincorporation of nucleotides into DNA, the supply of dNTPs supply can allow for DNA repair. p53R2 is a small subunit of ribonucleotide reductase that can induce such repair. Changes within this p53 induced R2 homolog can cause depletion in mitochondrial DNA and consequently p53R2 serves a major factor in dNTP supply.

RNR may use the morpheein model of allosteric regulation.

Clinical significance

Cancer

Ribonucleotide reductase (RNR) is a key enzyme responsible for the de novo synthesis of deoxyribonucleotides, which are essential for DNA replication and repair in all living cells. Its clinical significance arises from its central role in cell proliferation and genomic integrity making it a critical factor in cancer biology. In oncology, RNR activity is frequently elevated in malignant cells, where it supports rapid DNA synthesis and tumor growth. Overexpression of specific RNR subunits, such as RRM2, has been correlated with poor prognosis, increased chemoresistance, and unfavorable outcomes in various cancers, including pancreatic cancer and chronic lymphocytic leukemia. The prognostic value of RNR expression, particularly the RRM1 subunit, has been explored as both a biomarker and a potential target for targeted therapies.

Therapeutically, RNR is an established target for anticancer drugs—agents such as gemcitabine and hydroxyurea exert their effects by inhibiting RNR, thereby disrupting DNA synthesis and compromising cancer cell viability. Novel inhibitors and strategies aiming at RNR and its subunits continue to be investigated to overcome drug resistance and enhance cancer treatment outcomes. Furthermore, the enzyme's crucial function in maintaining the balance of deoxyribonucleotide pools positions it as a target not only for cancer therapy but also for antimicrobial and antiviral drug development, given its essentiality in prokaryotes and some viruses. If you can stop RNR, you can halt cancer progression. Therefore, this enzyme is considered a classical target for cancer therapeutics.

Inhibitors

Generally Class I RNR inhibitors can be divided in three main groups: translation inhibitors, which block the synthesis of the enzyme; dimerization inhibitors that prevent the association of the two RNR subunits (R1 and R2); and catalytic inhibitors that inactivate the subunit R1 and/or subunit R2. Although the C-terminus of RNR2 proteins is different across species, RNR2 can interact with RNR1 across species. When the mouse RNR2 C-terminus was replaced with the E. coli RNR2 C-terminal (7 or 33) amino acid residues, the chimeric RNR2 subunit still binds to mouse RNR1 subunits. However, they lack enzymatic activity due probably to the elimination of residues involved in the transfer of the free radical electron from the RNR2 to the RNR1 subunit. This inhibition RNR2 binding to RNR1 has been tested successfully in herpes simplex virus (HSV) RNR. When a 7 amino acid oligomer (GAVVNDL) truncated from the C-terminus of the RNR2 subunit was used in competition assays, it prevented the normal RNR2 from forming an enzymatically active complex with RNR1. Other small peptide inhibitors similar to the RNR2 C-terminus have also been used successfully to inhibit HSV RNR enzymatic activity and thus HSV replication. In mice models of stromal keratitis and corneal neovascularization (HSV ocular disease), a small RNR2 C-terminal analog BILD 1263 has been reported to inhibit RNR and is effective in preventing these diseases. In some cases, although treatment with small C-terminal analogs may not stop disease spreading, they can still help in healing. In the acyclovir-resistant HSV (PAAr5), a small peptide inhibitor BILD 1633 has been reported to be 5 to 10 times more potent than BILD 1263 against cutaneous PAAr5 infection. A combination therapy approach (BILD 1633 and acyclovir) is more effective to heal topical lesions in mice. These data suggest that small peptide inhibitors that compete with RNR2 for binding to RNR1 are useful in preventing the spread of HSV.

Gallium inhibits RNR2 by substituting for Fe<sup>3+</sup> in the active site. Gallium maltolate is an orally bioavailable form of gallium that exploits this inhibitory activity to treat cancer, infections, and other diseases.

The drugs hydroxyurea and Motexafin gadolinium interfere with the action of this enzyme.

References

  • The ribonucleotide reductase database (RNRdb)

it:Ribonucleoside-trifosfato reduttasi