Ribonucleotide reductase

Ribonucleotide reductase (RNR), also known as ribonucleoside diphosphate reductase (rNDP), is an

Each Class I alpha monomer consists of three domains:^[9]

• 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.

The Class I beta subunit usually contains a di-metal center and a stable

In A. aegypti mosquitoes, RNR1 retains most of the crucial amino acid residues, including aspartate (D64) and valine (V292 or V284), that are necessary in allosteric regulation; proline (P210 and P610), leucine (L453 and L473), and methionine (M603) residues that are located in the hydrophobic active site; cysteine (C225, C436 and C451) residues that are involved in removal of a hydrogen atom and transfer of the radical electron at the active site; cysteine (C225 and C436), asparagine (N434), and glutamate (E441) residues that bind the ribonucleotide substrate; tyrosine (Y723 and Y743) residues that dictate the radical transfer; and cysteine (C838 and C841) residues that are used in the regeneration of dithiol groups in the active site.^[14]

Function

The enzyme ribonucleotide reductase (RNR) catalyzes the de novo synthesis of dNDPs.^[16] Catalysis of ribonucleoside 5’-diphosphates (NDPs) involves a reduction at the 2’-carbon of ribose 5-phosphate to form the 2’-deoxy derivative-reduced 2’-deoxyribonucleoside 5’-diphosphates (dNDPs). This reduction is initiated with the generation of a free radical. Following a single reduction, RNR requires electrons donated from the dithiol groups of the protein thioredoxin. Regeneration of thioredoxin occurs when nicotinamide adenine dinucleotide phosphate (NADPH) provides two hydrogen atoms that are used to reduce the disulfide groups of thioredoxin.

Three classes of RNR have similar mechanisms for the reduction of NDPs, but differ in the domain that generates the free radical, the specific metal in the

The mechanism that is currently accepted for the reduction of ribonucleotides to deoxyribonucleotides is depicted in the following scheme. The first step involves the abstraction of the 3’- H of substrate 1 by radical Cys439. Subsequently, the reaction involves the elimination of one water molecule from carbon C-2’ of the ribonucleotide, catalyzed by Cys225 and Glu441. In the third step there is a hydrogen atom transfer from Cys225 to carbon C-2’ of the 2’-ketyl radical 3, after previous proton transfer from Cys462 to Cys225. At the end of this step, a radical anionic disulfide bridge and the closed-shell ketone intermediate 4 are obtained. This intermediate has been identified during the conversion of several 2’-substituted substrate analogues, as well as with the natural substrate^[17] 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..^[18][19]

Regulation

Class I RNR comprises RNR1 and RNR2 subunits, which can associate to form a heterodimeric tetramer.^[5] RNR1 contains both allosteric sites, mediating regulation of substrate specificity and activity.^[11] Depending on the allosteric configuration, one of the four ribonucleotides binds to the active site.

Regulation of RNR is designed to maintain balanced quantities of dNTPs. Binding of effector molecules either increases or decreases RNR activity. When ATP binds to the allosteric activity site, it activates RNR. In contrast, when dATP binds to this site, it deactivates RNR.^[9] In addition to controlling activity, the allosteric mechanism also regulates the substrate specificity and ensures the enzyme produces an equal amount of each dNTP for DNA synthesis.^[9] In all classes, binding of ATP or dATP to the allosteric site induces reduction of cytidine 5’-diphosphate (CDP) and uridine 5’-diphosphate (UDP); 2’-deoxyguanosine 5’-triphosphate (dGTP) induces reduction of adenosine 5’-diphosphate (ADP); and 2’-deoxythymidine 5’-triphosphate (dTTP) induces reduction of guanosine 5’-diphosphate (GDP) (Figure 1).

Class IB reductases are not inhibited by dATP because they lack approximately 50 N-terminal amino acids required for the allosteric activity site.^[20] 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.^[21] 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.^[22][23] 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.^[24]

RNR may use the morpheein model of allosteric regulation.^[25]

RNR1 and RNR2 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.^[18]

Class I RNR can be inhibited by peptides similar to the C-terminus of RNR2. These peptides can compete with RNR2 for binding to RNR1, and as a result RNR1 does not form an enzymatically active complex with RNR2.^[26][27] Although the C-terminus of RNR2 proteins is different across species, RNR2 can interact with RNR1 across species.^[28] 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.^[27]

Small peptides can specifically inhibit the RNR2 subunits from binding with RNR1 when they share a significant similarity with the normal RNR2 C-terminus.

Gallium inhibits RNR2 by substituting for Fe³⁺ 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.^[34]

The drugs