Ribonucleotide reductase

Ribonucleotide reductase (RNR, also known as ribonucleoside diphosphate reductase) is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. 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 synthesised by another enzyme (thymidylate kinase) from dTMP (deoxythymidine monophosphate).

Structure
The iron-dependent enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis. Class I RNR enzymes are constructed from large RNR1 and small RNR2 subunits which associate to form an active heterodimeric tetramer. Since the enzyme catalyses the de novo synthesis of deoxyribonucleotides (dNTPs), precursors to DNA synthesis, it is essential for cell proliferation.

In humans, the RNR1 subunit is encoded by the RRM1 gene while there are two isoforms of the RNR2 subunit, encoded by the RRM2 and RRM2B genes:

Each RNR1 monomer consists of three domains: In Pfam, the second domain has been interpreted as two separate 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.
 * a shorter all-alpha N-terminal domain,
 * and a longer barrel C-terminal domain.

RNR2 contains a diferric iron center and a stable tyrosyl radical. 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 [ aspartate (D146), glutamate (E177, E240, and E274) ] and two histidines (H180 and H277). Association occurs between the C-terminus of RNR2 and the C-terminus of RNR1. Enzymatic activity is dependent on association of the RNR1 and RNR2 subunits. The active site consists of the active dithiol groups from the RNR1 as well as the diferric center and the tyrosyl radical from the RNR2 subunit. Other residues of RNR2, such as aspartate (D273), tryptophan (W48), and tyrosine (Y356) further stabilize the active-site tyrosyl radical thus allowing electron transfer. These residues help in the transfer of the radical electron from tyrosine (Y122) of RNR2 to cysteine (C439) of RNR1. The electron transfer begins on RNR2 tyrosine (Y122) and continues in RNR2 to tryptophan (W48), which is separated from RNR1 tyrosine (Y731) by 2.5 nanometers. Electron transfer from RNR2 to RNR1 occurs via tyrosine (Y356 to Y731) and continues on through tyrosine (Y730) to cysteine (C439) in the active site. Site-directed mutations of the RNR primary structure indicate that all residues cited above participate in the long distance transfer of the free radical to the active site.

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.

Function


The enzyme ribonucleotide reductase (RNR) catalyzes the de novo synthesis of dNTPs. 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 metalloprotein structure, and the electron donors. All classes use free-radical chemistry. Class I reductases use an iron center with ferrous to ferric conversion to generate a tyrosyl free radical. Reduction of NDP substrates occurs under aerobic conditions. Class I reductases are divided into IA and IB due to differences in regulation. Class IA reductases are distributed in eukaryotes, eubacteria, bacteriophages, and viruses. Class IB reductases are found in eubacteria. Class IB reductases can also use a radical generated with the stabilization of a binuclear manganese center. Class II reductases generate a free radical by mechanisms involving 5’-deoxyadenosyl cobalamin (coenzyme B12) and have a simpler structure than class I and class III reductases. Reduction of NDPs or ribonucleotide 5’-triphosphates (NTPs) occurs under either aerobic or anaerobic conditions. Class II reductases are distributed in archaebacteria, eubacteria, and bacteriophages. Class III reductases use a glycine radical generated with the help of an S-adenosyl methionine and an iron sulphur center. Reduction of NTPs is limited to anaerobic conditions. Class III reductases are distributed in archaebacteria, eubacteria, and bacteriophages. Organisms are not limited to having one class of enzymes. For example, E. coli have both class I and class III RNR.

Metabolic pathways
Several major pathways lead to the generation of precursors for the de novo synthesis of nucleotides. These pathways involve the generation of ribose 5-phosphate, carbon dioxide, amino acids and ammonia. Ribose 5-phosphate generation begins with a molecule of glucose that is oxidized via the pentose phosphate pathway. The pentose phosphate pathway produces NADPH for reducing power involved in the catalysis of NTPs to dNTPs, and to produce ribose 5-phosphate necessary for the synthesis of ribonucleotides. Carbon dioxide is always available for biosynthesis because its concentration in the blood is kept nearly constant via the bicarbonate buffer system. An important co-factor for ribonucleotide synthesis is tetrahydrofolate, which is the major mediator for carbon transfers. Its derivative, folate (a vitamin), cannot be synthesized in mammals. Many forms of tetrahydrofolate follow pathways that are interconnected. For ribonucleotide synthesis, the N10-formyl-tetrahydrofolate molecule is necessary for the transfer of formyl groups to the purine ring. Amino groups or ammonia are donated from the catabolism of amino acids beginning with a dietary protein molecule. The free ammonia is combined with glutamate by a reaction involving adenosine 5’-triphosphate (ATP) and the activity of glutamine synthetase, which produces a nontoxic molecule of glutamine that can be transported in the bloodstream. Glutamine synthetase is present in nearly all organisms and is allosterically regulated by end products of glutamine metabolism. During synthesis of purines, amino groups are removed from glutamine for purine rings. Purine ribonucleotides are attached to ribose 5-phosphate during assembly of intermediate inosinate (IMP) from precursors in the purine pathway, including glutamine, glycine, N10-formyl-tetrahydrofolate, bicarbonate, aspartate and ATP. Synthesis is catalyzed by large multienzyme complexes. Purine ribonucleotides are adenosine 5’-monophosphate (AMP) and guanosine 5’-monophosphate (GMP). AMP is formed from IMP by aspartate donating an amino group (leaving as fumarate) and guanosine 5’-triphosphate (GTP) providing a phosphate. GMP is formed by the oxidation of IMP at C-2 requiring NAD+. Following oxidation, glutamine donates an amino group (leaving as glutamate) then ATP provides a phosphate.

Pyrimidine ribonucleotides are formed from an orotate molecule that is assembled from aspartate to form the pyrimidine ring. Subsequently, orotate is attached to ribose 5-phosphate to yield orotidylate. These two steps are catalyzed by a large multienzyme complex (CAD). Pyrimidine ribonucleotides are cytidine 5’-monophosphate (CMP) and uridine 5’-monophosphate (UMP). Orotidylate is decarboxylated to form UMP. UMP and two ATPs are transferred by kinases to form uridine 5’-triphosphate (UTP). Cytidine 5’-triphosphate (CTP) is formed from UTP by glutamine donating an amino group (leaving as glutamate) and ATP providing a phosphate. In some species, ammonia can donate an amino group instead of glutamine.

Generation of 2’-deoxythymidine 5’-monophosphate (dTMP) occurs by conversion of 2’-deoxyuridine 5’-monophosphate (dUMP). Thymidylate synthase catalyzes the reaction in which dTMP is formed from dUMP; to provide the carbon atom N5, N10-methylene-tetrahydrofolate is oxidized to 7, 8-dihydrofolate. Dihydrofolate reductase (DHFR) is an essential enzyme that regenerates tetrahydrofolate at the expense of NADPH. Ribonucleoside monophosphates (AMP, GMP, CMP, and UMP) are phosphorylated to ribonucleoside diphosphates for their particular base by specific kinases. Ribonucleoside diphosphates are phosphorylated a second time to ribonucleoside triphosphates by nucleoside-diphosphate kinase, which is not specific for their base or for their 2’-carbon of ribose 5-phosphate and its 2’-deoxy derivative. The activity of nucleoside diphosphate kinase is sequential based on which class of RNR is used. These metabolic pathways generate the ribonucleotides (ATP, GTP, CTP, and UTP) that are precursors for dNTPs. Thus, RNR reduces the corresponding NTPs to dNTPs for DNA synthesis. Cellular concentration of dNTP is much lower than required for DNA replication, and RNR is essential for adequate precursors during DNA synthesis.

After RNR reduces NDP or NTP the enzyme becomes inactive because a disulfide bond is formed in the active site. An exchange reaction occurs that reduces the disulfide bond of RNR catalyzed by thioredoxin or glutaredoxin. RNR gains electrons on the active-site dithiol groups necessary for its activity.

Catalytic Reduction Mechanism
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 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


Class I RNR comprises RNR1 and RNR2 subunits, which can associate to form a heterodimeric tetramer. RNR1 contains both allosteric sites, mediating regulation of substrate specificity and activity. 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. 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. 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). Interestingly, class IB reductases are not inhibited by dATP because they lack approximately 50 N-terminal amino acids required for the allosteric activity site. 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.

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.

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. 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.

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

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