Ribonucleotide reductase (RNR), also known as ribonucleoside diphosphate reductase (rNDP), is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
that catalyzes the formation of
deoxyribonucleotide
A deoxyribonucleotide is a nucleotide that contains deoxyribose. They are the monomeric units of the informational biopolymer, deoxyribonucleic acid ( DNA). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar (monosaccharide), a nit ...
s from
ribonucleotide
In biochemistry, a ribonucleotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic ...
s.
It catalyzes this formation by removing the 2'-hydroxyl group of the ribose ring of nucleoside diphosphates. 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
Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there ar ...
and
DNA repair
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...
.
A somewhat unusual feature of the RNR enzyme is that it catalyzes a reaction that proceeds via a
free radical
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Ageing
Ailments of unknown cause
Biogerontology
Biological processes
Causes of death
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Life extension
Metabo ...
mechanism of action.
The substrates for RNR are
ADP,
GDP
Gross domestic product (GDP) is a monetary measure of the market value of all the final goods and services produced and sold (not resold) in a specific time period by countries. Due to its complex and subjective nature this measure is ofte ...
,
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
In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' has ...
tetramer
A tetramer () ('' tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula ...
. By reducing NDPs to 2'-dNDPs, the enzyme catalyses the
de novo synthesis
In chemistry, ''de novo'' synthesis () refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to recycling after partial degradation. For example, nucleotides are not needed in the diet as ...
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:
Each Class I alpha
monomer
In chemistry, a monomer ( ; '' mono-'', "one" + ''-mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization.
Classification
...
consists of three
domain
Domain may refer to:
Mathematics
*Domain of a function, the set of input values for which the (total) function is defined
**Domain of definition of a partial function
**Natural domain of a partial function
**Domain of holomorphy of a function
* Do ...
s:
* 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
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
aspartic_acid">aspartate_(D146),_ aspartic_acid">aspartate_(D146),_glutamic_acid">glutamate_(E177,_E240,_and_E274).html" ;"title="glutamic_acid.html" ;"title="aspartic_acid.html" ;"title="/nowiki>aspartic acid">aspartate (D146), glutamic acid">glutamate (E177, E240, and E274)">glutamic_acid.html" ;"title="aspartic_acid.html" ;"title="/nowiki>aspartic acid">aspartate (D146), glutamic acid">glutamate (E177, E240, and E274)/nowiki> 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
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
(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
Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...
(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
Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...
(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 dNDPs. Catalysis of ribonucleoside 5’-diphosphates (NDPs) involves a reduction at the 2’-carbon of ribose 5-phosphate
Ribose 5-phosphate (R5P) is both a product and an intermediate of the pentose phosphate pathway. The last step of the oxidative reactions in the pentose phosphate pathway is the production of ribulose 5-phosphate. Depending on the body's state, r ...
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
Thioredoxin is a class of small redox proteins known to be present in all organisms. It plays a role in many important biological processes, including redox signaling. In humans, thioredoxins are encoded by ''TXN'' and '' TXN2'' genes. Loss-of-fu ...
. Regeneration of thioredoxin occurs when nicotinamide adenine dinucleotide phosphate ( NADPH) provides two hydrogen atoms that are used to reduce the disulfide
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
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
Metalloprotein is a generic term for a protein that contains a metal ion cofactor. A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins (out of ~20,000) contain zinc-binding protein domains al ...
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
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were amon ...
, bacteriophages, and virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsk ...
es. Class IB reductases are found in eubacteria. Class IB reductases can also use a radical generated with the stabilization of a binuclear manganese
Manganese is a chemical element with the symbol Mn and atomic number 25. It is a hard, brittle, silvery metal, often found in minerals in combination with iron. Manganese is a transition metal with a multifaceted array of industrial alloy use ...
center. Class II reductases generate the free radical 5’-deoxyadenosyl radical from cobalamin
Vitamin B12, also known as cobalamin, is a water-soluble vitamin involved in metabolism. It is one of eight B vitamins. It is required by animals, which use it as a cofactor in DNA synthesis, in both fatty acid and amino acid metabolism. ...
(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
Aerobic means "requiring air," in which "air" usually means oxygen.
Aerobic may also refer to
* Aerobic exercise, prolonged exercise of moderate intensity
* Aerobics, a form of aerobic exercise
* Aerobic respiration, the aerobic process of cel ...
or anaerobic conditions. Class II reductases are distributed in archaebacteria
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebact ...
, eubacteria, and bacteriophages. Class III reductases use a glycine radical generated with the help of an S-adenosyl methionine
''S''-Adenosyl methionine (SAM), also known under the commercial names of SAMe, SAM-e, or AdoMet, is a common cosubstrate involved in methyl group transfers, transsulfuration, and aminopropylation. Although these anabolic reactions occur throug ...
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.
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).
Class IB reductases are not inhibited by dATP because they lack approximately 50 N-terminal amino acids required for the allosteric activity site. 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.
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 peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
...
s 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
Keratitis is a condition in which the eye's cornea, the clear dome on the front surface of the eye, becomes inflamed. The condition is often marked by moderate to intense pain and usually involves any of the following symptoms: pain, impaired e ...
and corneal neovascularization
Corneal neovascularization (CNV) is the in-growth of new blood vessels from the pericorneal plexus into avascular corneal tissue as a result of oxygen deprivation. Maintaining avascularity of the corneal stroma is an important aspect of corneal ...
( 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
Aciclovir (ACV), also known as acyclovir, is an antiviral medication. It is primarily used for the treatment of herpes simplex virus infections, chickenpox, and shingles. Other uses include prevention of cytomegalovirus infections following t ...
-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
Gallium maltolate is a coordination complex consisting of a trivalent gallium cation coordinated to three maltolate ligands. The compound is a potential therapeutic agent for cancer, infectious disease, and inflammatory disease. A cosmetic skin ...
is an orally bioavailable form of gallium that exploits this inhibitory activity to treat cancer, infections, and other diseases.
The drugs hydroxyurea
Hydroxycarbamide, also known as hydroxyurea, is a medication used in sickle-cell disease, essential thrombocythemia, chronic myelogenous leukemia, polycythemia vera, and cervical cancer. In sickle-cell disease it increases fetal hemoglobin and d ...
and Motexafin gadolinium
Motexafin gadolinium (proposed tradename Xcytrin) is an inhibitor of thioredoxin reductase and ribonucleotide reductase. It has been proposed as a possible chemotherapeutic agent in the treatment of brain metastases.
History
On May 9, 2006, a Ne ...
interfere with the action of this enzyme.
References
External links
*
The ribonucleotide reductase database (RNRdb)
{{Portal bar, Biology, border=no
EC 1.17.4
it:Ribonucleoside-trifosfato reduttasi