View source for Ribonucleotide reductase

{{Short description|Class of enzymes}}
{{infobox enzyme
| Name       = ribonucleoside-diphosphate reductase
| EC_number  = 1.17.4.1
| CAS_number = 9047-64-7sy
| GO_code    = 0004748
| image      = 3uus.jpg
| width      = 270
| caption    = Ribonucleoside-diphosphate reductase heterooctamer, E.Coli
}}

'''Ribonucleotide reductase''' ('''RNR'''), also known as '''ribonucleoside diphosphate reductase''', is an [[enzyme]] that catalyzes the formation of [[deoxyribonucleotide]]s from [[ribonucleotide]]s.<ref name="pmid22050358">{{cite journal | vauthors = Hofer A, Crona M, Logan DT, Sjöberg BM | title = DNA building blocks: keeping control of manufacture | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 47 | issue = 1 | pages = 50–63 | date = 2012 | pmid = 22050358 | doi = 10.3109/10409238.2011.630372 | doi-access = free | pmc = 3267527 }}</ref><ref name="pmid1412696">{{cite journal | vauthors = Elledge SJ, Zhou Z, Allen JB | title = Ribonucleotide reductase: regulation, regulation, regulation | journal = Trends in Biochemical Sciences | volume = 17 | issue = 3 | pages = 119–23 | date = March 1992 | pmid = 1412696 | doi = 10.1016/0968-0004(92)90249-9 }}</ref> 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.<ref name="pmid15262976">{{cite journal | vauthors = Sneeden JL, Loeb LA | title = Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea | journal = The Journal of Biological Chemistry | volume = 279 | issue = 39 | pages = 40723–8 | date = September 2004 | pmid = 15262976 | doi = 10.1074/jbc.M402699200 | doi-access = free }}</ref> Deoxyribonucleotides in turn are used in the synthesis of [[deoxyribonucleic acid|DNA]]. The reaction catalyzed by RNR is strictly conserved in all living organisms.<ref name="pmid12107591">{{cite journal | vauthors = Torrents E, Aloy P, Gibert I, Rodríguez-Trelles F | title = Ribonucleotide reductases: divergent evolution of an ancient enzyme | journal = Journal of Molecular Evolution | volume = 55 | issue = 2 | pages = 138–52 | date = August 2002 | pmid = 12107591 | doi = 10.1007/s00239-002-2311-7 | bibcode = 2002JMolE..55..138T | s2cid = 24603578 | doi-access = free }}</ref> 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]].<ref name="pmid17229208">{{cite journal | vauthors = Herrick J, Sclavi B | title = Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage | journal = Molecular Microbiology | volume = 63 | issue = 1 | pages = 22–34 | date = January 2007 | pmid = 17229208 | doi = 10.1111/j.1365-2958.2006.05493.x | s2cid = 9473163 | doi-access = free }}</ref> A somewhat unusual feature of the RNR enzyme is that it catalyzes a reaction that proceeds via a [[free radical]] mechanism of action.<ref name="Eklund_1997">{{cite journal | vauthors = Eklund H, Eriksson M, Uhlin U, Nordlund P, Logan D | title = Ribonucleotide reductase—structural studies of a radical enzyme | journal = Biological Chemistry | volume = 378 | issue = 8 | pages = 821–5 | date = August 1997 | pmid = 9377477 | doi = 10.1515/bchm.1997.378.8.815 }}</ref><ref name="pmid9852763">{{cite journal | vauthors = Stubbe J, Riggs-Gelasco P | title = Harnessing free radicals: formation and function of the tyrosyl radical in ribonucleotide reductase | journal = Trends in Biochemical Sciences | volume = 23 | issue = 11 | pages = 438–43 | date = November 1998 | pmid = 9852763 | doi = 10.1016/S0968-0004(98)01296-1 }}</ref> The substrates for RNR are [[adenosine diphosphate|ADP]], [[guanosine diphosphate|GDP]], [[cytidine diphosphate|CDP]] and [[uridine diphosphate|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]] [[Tetrameric protein|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 growth|cell proliferation]].<ref name="pmid21336276">{{cite journal | vauthors = Fairman JW, Wijerathna SR, Ahmad MF, Xu H, Nakano R, Jha S, Prendergast J, Welin RM, Flodin S, Roos A, Nordlund P, Li Z, Walz T, Dealwis CG | title = Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization | journal = Nature Structural & Molecular Biology | volume = 18 | issue = 3 | pages = 316–22 | date = March 2011 | pmid = 21336276 | pmc = 3101628 | doi = 10.1038/nsmb.2007 }}</ref> 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.<ref name="pmid15475969">{{cite journal | vauthors = Larsson KM, Jordan A, Eliasson R, Reichard P, Logan DT, Nordlund P | title = Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase | journal = Nature Structural & Molecular Biology | volume = 11 | issue = 11 | pages = 1142–9 | date = November 2004 | pmid = 15475969 | doi = 10.1038/nsmb838 | s2cid = 1025702 }}</ref>

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:
{|
|- valign=top
|{{infobox protein
| Name = [[RRM1|ribonucleotide reductase M1 polypeptide]]
| caption = 
| image = 
| width = 
| HGNCid = 10451
| Symbol = [[RRM1]]
| AltSymbols = 
| EntrezGene = 6240
| OMIM = 180410
| RefSeq = NM_001033
| UniProt = P23921
| PDB = 
| ECnumber = 1.17.4.1
| Chromosome = 11
| Arm = p
| Band = 15.5–15.4
| LocusSupplementaryData = 
}}
|{{infobox protein
| Name = [[RRM2|ribonucleotide reductase M2 polypeptide]]
| caption = 
| image = 
| width = 
| HGNCid = 10452
| Symbol = [[RRM2]]
| AltSymbols = 
| EntrezGene = 6241
| OMIM = 180390
| RefSeq = NM_001034
| UniProt = P31350
| PDB = 
| ECnumber = 1.17.4.1
| Chromosome = 2
| Arm = p
| Band = 25
| LocusSupplementaryData = -p24
}}
|{{infobox protein
| Name = [[RRM2B|ribonucleotide reductase M2 B]] (TP53 inducible)
| caption = 
| image = 
| width = 
| HGNCid = 17296
| Symbol = [[RRM2B]]
| AltSymbols = 
| EntrezGene = 50484
| OMIM = 604712
| RefSeq = NM_015713
| UniProt = Q9NTD8
| PDB = 
| ECnumber = EC:1.17.4.1
| Chromosome = 8
| Arm = q
| Band = 23.1
| LocusSupplementaryData = 
}}
|}

Each Class I alpha [[monomer]] consists of three [[protein domain|domain]]s:<ref name="Jordan_Reichard_1998">{{cite journal | vauthors = Jordan A, Reichard P | title = Ribonucleotide reductases | journal = Annual Review of Biochemistry | volume = 67 | issue = 1 | pages = 71–98 | year = 1998 | pmid = 9759483 | doi = 10.1146/annurev.biochem.67.1.71 | doi-access = free }}</ref>
* one mainly helical domain comprising the 220 [[N-terminus|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.

{|
|- valign=top
|{{Infobox protein family
| Symbol = RR_N
| Name = Ribonucleotide reductase<br />N-terminal
| image = 1PEU_R1E.png
| width = 
| caption = Crystallographic structure of the ribonucleotide reductase protein R1E from ''[[Salmonella typhimurium|S. typhimurium]]''.  The protein is rainbow colored ([[N-terminus]] = blue, [[C-terminus]] = red) while [[deoxyadenosine triphosphate|dATP]] is depicted as sticks and a complexed [[magnesium in biology|magnesium]] ion as a grey sphere.<ref name="pmid12818204">{{PDB|1PEU}}; {{cite journal | vauthors = Uppsten M, Färnegårdh M, Jordan A, Eliasson R, Eklund H, Uhlin U | title = Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors | journal = Journal of Molecular Biology | volume = 330 | issue = 1 | pages = 87–97 | date = June 2003 | pmid = 12818204 | doi = 10.1016/S0022-2836(03)00538-2 }}</ref>
| Pfam = PF08343
| InterPro = IPR013554
| SMART = 
| PROSITE = 
| SCOP = 1peq
| TCDB = 
| OPM family = 
| OPM protein = 
| PDB = {{PDB2|1pem}}, {{PDB2|1peo}}, {{PDB2|1peq}}, {{PDB2|1peu}}, {{PDB2|2bq1}}
}}
|{{Infobox protein family
| Symbol = Ribonuc_red_lgN
| Name = Ribonucleotide reductase<br />all-alpha domain
| image = PDB 1rlr EBI.jpg
| width = 
| caption = Structure of ribonucleotide reductase protein R1 (Class I alpha subunit).<ref name="pmid8052308">{{cite journal | vauthors = Uhlin U, Eklund H | title = Structure of ribonucleotide reductase protein R1 | journal = Nature | volume = 370 | issue = 6490 | pages = 533–9 | date = August 1994 | pmid = 8052308 | doi = 10.1038/370533a0 | bibcode = 1994Natur.370..533U | s2cid = 8940689 }}</ref>
| Pfam = PF00317
| InterPro = IPR013509
| SMART = 
| PROSITE = PDOC00084
| SCOP = 1rlr
| TCDB = 
| OPM family = 
| OPM protein = 
| PDB = {{PDB2|1pem}}, {{PDB2|1peo}}, {{PDB2|1peq}}, {{PDB2|1peu}}, {{PDB2|1r1r}}, {{PDB2|2r1r}}, {{PDB2|3r1r}}, {{PDB2|4r1r}}, {{PDB2|5r1r}}, {{PDB2|6r1r}}, {{PDB2|7r1r}}
}}
|{{Infobox protein family
| Symbol = Ribonuc_red_lgC
| Name = Ribonucleotide reductase<br />barrel domain
| image = PDB 1pem EBI.jpg
| width = 
| caption = Structure of ribonucleotide reductase protein R1E from ''Salmonella typhimurium''.<ref name="pmid12818204"/>
| Pfam = PF02867
| Pfam_clan = CL0339
| InterPro = IPR000788
| SMART = 
| PROSITE = PDOC00084
| SCOP = 1rlr
| TCDB = 
| OPM family = 
| OPM protein = 
| PDB = {{PDB2|1pem}}, {{PDB2|1peo}}, {{PDB2|1peq}}, {{PDB2|1peu}}, {{PDB2|1r1r}}, {{PDB2|2r1r}}, {{PDB2|3r1r}}, {{PDB2|4r1r}}, {{PDB2|5r1r}}, {{PDB2|6r1r}}, {{PDB2|7r1r}}
}}
|{{Infobox protein family
| Symbol = Ribonuc_red_sm
| Name = Ribonucleotide reductase<br />small chain
| image = PDB 1rib EBI.jpg
| width = 
| caption = Structure of the ''Escherichia coli'' ribonucleotide reductase protein R2.<ref name="pmid8331655">{{cite journal | vauthors = Nordlund P, Eklund H | title = Structure and function of the Escherichia coli ribonucleotide reductase protein R2 | journal = Journal of Molecular Biology | volume = 232 | issue = 1 | pages = 123–64 | date = July 1993 | pmid = 8331655 | doi = 10.1006/jmbi.1993.1374 }}</ref>
| Pfam = PF00268
| Pfa,_clan = CL0044
| InterPro = IPR000358
| SMART = 
| PROSITE = PDOC00317
| SCOP = 1rib
| TCDB = 
| OPM family = 
| OPM protein = 
| PDB = {{PDB2|1av8}}, {{PDB2|1biq}}, {{PDB2|1h0n}}, {{PDB2|1h0o}}, {{PDB2|1jk0}}, {{PDB2|1jpr}}, {{PDB2|1jqc}}, {{PDB2|1kgn}}, {{PDB2|1kgo}}, {{PDB2|1kgp}}, {{PDB2|1mrr}}, {{PDB2|1mxr}}, {{PDB2|1oqu}}, {{PDB2|1pfr}}, {{PDB2|1pim}}, {{PDB2|1piu}}, {{PDB2|1piy}}, {{PDB2|1piz}}, {{PDB2|1pj0}}, {{PDB2|1pj1}}, {{PDB2|1pm2}}, {{PDB2|1r2f}}, {{PDB2|1r65}}, {{PDB2|1rib}}, {{PDB2|1rnr}}, {{PDB2|1rsr}}, {{PDB2|1rsv}}, {{PDB2|1smq}}, {{PDB2|1sms}}, {{PDB2|1syy}}, {{PDB2|1uzr}}, {{PDB2|1w68}}, {{PDB2|1w69}}, {{PDB2|1xik}}, {{PDB2|1xsm}}, {{PDB2|1yfd}}, {{PDB2|2alx}}, {{PDB2|2av8}}, {{PDB2|2bq1}}, {{PDB2|2r2f}}
}}
|}
The Class I beta subunit usually contains a di-metal center and a stable [[tyrosine|tyrosyl]] [[free radical|radical]]. In humans, the beta subunit relies on a di-iron cofactor.  In ''[[Escherichia coli|E. coli]]'', the tyrosyl radical is located at position 122 (Y122) providing the stable radical for the Class I RNR2 subunits.<ref name="Högbom_2001">{{cite journal | vauthors = Högbom M, Andersson ME, Nordlund P | title = Crystal structures of oxidized dinuclear manganese centres in Mn-substituted class I ribonucleotide reductase from Escherichia coli: carboxylate shifts with implications for O2 activation and radical generation | journal = Journal of Biological Inorganic Chemistry | volume = 6 | issue = 3 | pages = 315–23 | date = March 2001 | pmid = 11315567 | doi = 10.1007/s007750000205 | s2cid = 20748553 }}</ref> In ''[[Aedes aegypti|A. aegypti]]'', this tyrosyl radical is located at position 184 (Y184).<ref name="Pham_2002">{{cite journal | vauthors = Pham DQ, Blachuta BJ, Nichol H, Winzerling JJ | title = Ribonucleotide reductase subunits from the yellow fever mosquito, Aedes aegypti: cloning and expression | journal = Insect Biochemistry and Molecular Biology | volume = 32 | issue = 9 | pages = 1037–44 | date = September 2002 | pmid = 12213240 | doi = 10.1016/S0965-1748(02)00041-3 | bibcode = 2002IBMB...32.1037P }}</ref> 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>[[aspartic acid|aspartate]] (D146), [[glutamic acid|glutamate]] (E177, E240, and E274)<nowiki>]</nowiki> and two [[histidine]]s (H180 and H277).<ref name="Pham_2002"/> Association occurs between the C-terminus of RNR2 and the [[C-terminus]] of RNR1.<ref name="Jordan_Reichard_1998"/> 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.<ref name="Jordan_Reichard_1998"/> 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 [[nanometer]]s. 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.<ref name="Chang_2004">{{cite journal | vauthors = Chang MC, Yee CS, Stubbe J, Nocera DG | title = Turning on ribonucleotide reductase by light-initiated amino acid radical generation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 101 | issue = 18 | pages = 6882–7 | date = May 2004 | pmid = 15123822 | pmc = 406436 | doi = 10.1073/pnas.0401718101 | bibcode = 2004PNAS..101.6882C | doi-access = free }}</ref> 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.<ref name="Jordan_Reichard_1998"/>

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.<ref name="Pham_2002"/>

The [[yeast]] ''[[Saccharomyces cerevisiae]]'' possesses a minor [[Protein isoform|isoform]] of large subunit of [[Ribonucleoside-diphosphate reductase|ribonucleotide-diphosphate reductase]] under the designation '''RNR3''' or ''YIL066C'' on the yeast chromosome IX.<ref name="entrez">{{cite web | title = RNR3 ribonucleotide-diphosphate reductase subunit RNR3 [Saccharomyces cerevisiae S288C] | url = https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=854744| accessdate = 2024-08-01}}</ref> It is a paralog of the yeast '''RNR1''', which likely arose from a whole [[genome duplication]] event.<ref>{{Cite web|url=https://www.yeastgenome.org/locus/RNR3|title = Rnr3 &#124; SGD}}</ref> The [[RNP complex|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 ==
[[File:Ribonucleotide reductase mechanism.jpg|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 de novo synthesis of dNDPs.<ref name="isbn0-7167-7108-X">{{cite book | last1 = Cox | first1 = Michael | last2 = Nelson | first2 = David R. | name-list-style = vanc | title = Lehninger Principles of Biochemistry | publisher = W. H. Freeman | location = San Francisco | year = 2008 | isbn = 978-0-7167-7108-1 | url = https://archive.org/details/lehningerprincip00lehn_1 | url-access = registration }}</ref> 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 ([[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.<ref name="Jordan_Reichard_1998"/> 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 [[eukaryote]]s, [[bacteria|eubacteria]], [[bacteriophage]]s, and [[virus]]es. 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 the free radical [[deoxyadenosyl radical|5’-deoxyadenosyl radical]] from [[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 [[Oxygen|aerobic]] or anaerobic conditions. Class II reductases are distributed in [[archaea|archaebacteria]], eubacteria, and bacteriophages. Class III reductases use a glycine radical generated with the help of an [[S-Adenosyl methionine|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.<ref name="Jordan_Reichard_1998"/><ref name="Pham_2002"/> 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 ==
[[Image:RNR reaction.png|thumb|left|400px|'''The reaction mechanism of RNR'''.]] 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<ref name="Cerqueira_2004">{{cite journal | vauthors = Cerqueira NM, Fernandes PA, Eriksson LA, Ramos MJ | title = Ribonucleotide activation by enzyme ribonucleotide reductase: understanding the role of the enzyme | journal = Journal of Computational Chemistry | volume = 25 | issue = 16 | pages = 2031–7 | date = December 2004 | pmid = 15481089 | doi = 10.1002/jcc.20127 | s2cid = 19665974 }}</ref> 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.''.<ref name="Cerqueira_2005">{{cite journal | vauthors = Cerqueira NM, Pereira S, Fernandes PA, Ramos MJ | title = Overview of ribonucleotide reductase inhibitors: an appealing target in anti-tumour therapy | journal = Current Medicinal Chemistry | volume = 12 | issue = 11 | pages = 1283–94 | year = 2005 | pmid = 15974997 | doi = 10.2174/0929867054020981 }}</ref><ref name="Cerqueira_2006">{{cite journal | vauthors = Cerqueira NM, Fernandes PA, Eriksson LA, Ramos MJ | title = Dehydration of ribonucleotides catalyzed by ribonucleotide reductase: the role of the enzyme | journal = Biophysical Journal | volume = 90 | issue = 6 | pages = 2109–19 | date = March 2006 | pmid = 16361339 | pmc = 1386789 | doi = 10.1529/biophysj.104.054627 | bibcode = 2006BpJ....90.2109C }}</ref>

== Regulation ==
[[File:Regulation of class-I ribonucleotide reductase.jpg|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.<ref name="Eklund_1997"/> RNR1 contains both allosteric sites, mediating regulation of substrate specificity and activity.<ref name="pmid8052308" /> 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.<ref name="Jordan_Reichard_1998"/> 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.<ref name="Jordan_Reichard_1998"/> 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.<ref name="Eliasson_1996">{{cite journal | vauthors = Eliasson R, Pontis E, Jordan A, Reichard P | title = Allosteric regulation of the third ribonucleotide reductase (NrdEF enzyme) from enterobacteriaceae | journal = The Journal of Biological Chemistry | volume = 271 | issue = 43 | pages = 26582–7 | date = October 1996 | pmid = 8900130 | doi = 10.1074/jbc.271.43.26582 | doi-access = free }}</ref> 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.<ref name="pmid17534360">{{cite journal | vauthors = Thelander L | title = Ribonucleotide reductase and mitochondrial DNA synthesis | journal = Nature Genetics | volume = 39 | issue = 6 | pages = 703–4 | date = June 2007 | pmid = 17534360 | doi = 10.1038/ng0607-703 | s2cid = 22565931 }}</ref>  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.<ref name="Kunz_1998">{{cite journal | vauthors = Kunz BA | title = Mutagenesis and deoxyribonucleotide pool imbalance | journal = Mutation Research | volume = 200 | issue = 1–2 | pages = 133–47 | year = 1988 | pmid = 3292903 | doi = 10.1016/0027-5107(88)90076-0 | bibcode = 1988MRFMM.200..133K }}</ref><ref name="Meuth_1998">{{cite journal | vauthors = Meuth M | title = The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells | journal = Experimental Cell Research | volume = 181 | issue = 2 | pages = 305–16 | date = April 1989 | pmid = 2647496 | doi = 10.1016/0014-4827(89)90090-6 }}</ref> 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.<ref name="pmid17486094">{{cite journal | vauthors = Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, Aubert S, Chrétien D, de Lonlay P, Paquis-Flucklinger V, Arakawa H, Nakamura Y, Munnich A, Rötig A | title = Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion | journal = Nature Genetics | volume = 39 | issue = 6 | pages = 776–80 | date = June 2007 | pmid = 17486094 | doi = 10.1038/ng2040 | s2cid = 22103978 }}</ref>

RNR may use the [[morpheein]] model of [[allosteric regulation]].<ref name=pmid22182754>{{cite journal | vauthors = Selwood T, Jaffe EK | title = Dynamic dissociating homo-oligomers and the control of protein function | journal = Archives of Biochemistry and Biophysics | volume = 519 | issue = 2 | pages = 131–43 | date = March 2012 | pmid = 22182754 | pmc = 3298769 | doi = 10.1016/j.abb.2011.11.020 }}</ref>

== Importance of RNR in Cancer Research ==
RNR is of paramount importance in cancer research since it is the rate-limiting enzyme in cancer growth. The activity of this enzyme is most closely associated, by far, with malignant cell growth. <ref>Weber G. Biochemical strategy of cancer cells and the design of chemotherapy: G. H. A. Clowes memorial lecture. Cancer Res. 1983;43(8):3466–92</ref> If you can stop RNR, you can halt cancer progression. Therefore, this enzyme is considered a classical target for cancer therapeutics. <ref>Shao J, Liu X, Zhu L, Yen Y. Targeting ribonucleotide reductase for cancer therapy. Expert Opin Ther Targets. 2013;17:1423–37. https://doi.org/10.1517/14728222.2013.840293. </ref> Chemotherapeutic compounds aiming to block this enzyme have been synthesized since the early 1970s. <ref>Agrawal KC, Booth BA, Sartorelli AC. Potential antitumor agents. 7. 4’-diethyleneoxy derivatives of -(N)-heterocyclic carboxaldehyde thiosemicarbazones. J Med Chem. 1973;16:715–7. https://doi.org/10.1021/jm00264a035.</ref>

== 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.<ref name="Cerqueira_2005"/>

Class I RNR can be inhibited by [[peptide]]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.<ref name="Climent_1991">{{cite journal | vauthors = Climent I, Sjöberg BM, Huang CY | title = Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies | journal = Biochemistry | volume = 30 | issue = 21 | pages = 5164–71 | date = May 1991 | pmid = 2036382 | doi = 10.1021/bi00235a008 }}</ref><ref name="Hamann_1998">{{cite journal | vauthors = Hamann CS, Lentainge S, Li LS, Salem JS, Yang FD, Cooperman BS | title = Chimeric small subunit inhibitors of mammalian ribonucleotide reductase: a dual function for the R2 C-terminus? | journal = Protein Engineering | volume = 11 | issue = 3 | pages = 219–24 | date = March 1998 | pmid = 9613846 | doi = 10.1093/protein/11.3.219 | doi-access = free }}</ref> Although the C-terminus of RNR2 proteins is different across species, RNR2 can interact with RNR1 across species.<ref name="Cosentino_1991">{{cite journal | vauthors = Cosentino G, Lavallée P, Rakhit S, Plante R, Gaudette Y, Lawetz C, Whitehead PW, Duceppe JS, Lépine-Frenette C, Dansereau N | title = Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit | journal = Biochemistry and Cell Biology | volume = 69 | issue = 1 | pages = 79–83 | date = January 1991 | pmid = 2043345 | doi = 10.1139/o91-011 }}</ref> 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.<ref name="Hamann_1998"/>

Small peptides can specifically inhibit the RNR2 subunits from binding with RNR1 when they share a significant similarity with the normal RNR2 C-terminus.<ref name="Cooperman_2003">{{cite journal | vauthors = Cooperman BS | title = Oligopeptide inhibition of class I ribonucleotide reductases | journal = Biopolymers | volume = 71 | issue = 2 | pages = 117–31 | year = 2003 | pmid = 12767114 | doi = 10.1002/bip.10397 | s2cid = 25196379 }}</ref> 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.<ref name="Filatov_1992">{{cite journal | vauthors = Filatov D, Ingemarson R, Gräslund A, Thelander L | title = The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure | journal = The Journal of Biological Chemistry | volume = 267 | issue = 22 | pages = 15816–22 | date = August 1992 | doi = 10.1016/S0021-9258(19)49608-7 | pmid = 1322407 | url = http://www.jbc.org/cgi/content/abstract/267/22/15816 | doi-access = free }}</ref> 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.<ref name="Cohen_1986">{{cite journal | vauthors = Cohen EA, Gaudreau P, Brazeau P, Langelier Y | title = Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2 | journal = Nature | volume = 321 | issue = 6068 | pages = 441–3 | year = 1986 | pmid = 3012360 | doi = 10.1038/321441a0 | bibcode = 1986Natur.321..441C | s2cid = 4238076 }}</ref> In mice models of [[stroma (animal tissue)|stromal]] [[keratitis]] and [[corneal neovascularization]] ([[herpes simplex|HSV]] [[eye disease|ocular disease]]), a small RNR2 C-terminal analog BILD 1263 has been reported to inhibit RNR and is effective in preventing these diseases.<ref name="Brandt_1996">{{cite journal | vauthors = Brandt CR, Spencer B, Imesch P, Garneau M, Déziel R | title = Evaluation of a peptidomimetic ribonucleotide reductase inhibitor with a murine model of herpes simplex virus type 1 ocular disease | journal = Antimicrobial Agents and Chemotherapy | volume = 40 | issue = 5 | pages = 1078–84 | date = May 1996 | pmid = 8723444 | pmc = 163269 | doi =  10.1128/aac.40.5.1078| url = }}</ref> 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.<ref name="Duan_1998">{{cite journal | vauthors = Duan J, Liuzzi M, Paris W, Lambert M, Lawetz C, Moss N, Jaramillo J, Gauthier J, Déziel R, Cordingley MG | title = Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo | journal = Antimicrobial Agents and Chemotherapy | volume = 42 | issue = 7 | pages = 1629–35 | date = July 1998 | pmid = 9660995 | pmc = 105657 | doi =  10.1128/aac.42.7.1629| url = }}</ref> 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.<ref name="PharmRev">{{cite journal | vauthors = Bernstein LR | title = Mechanisms of therapeutic activity for gallium | journal = Pharmacological Reviews | volume = 50 | issue = 4 | pages = 665–82 | date = December 1998 | pmid = 9860806 | url = http://pharmrev.aspetjournals.org/cgi/reprint/50/4/665.pdf }}</ref>

The drugs [[hydroxyurea]]<ref>{{cite web|title=Information on EC 1.17.4.1 – ribonucleoside-diphosphate reductase|url=http://www.brenda-enzymes.org/enzyme.php?ecno=1.17.4.1|website=Brenda|access-date=25 July 2015}}</ref> and [[Motexafin gadolinium]] interfere with the action of this enzyme.<ref name="Hashemy_2006">{{cite journal | vauthors = Hashemy SI, Ungerstedt JS, Zahedi Avval F, Holmgren A | title = Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase | journal = The Journal of Biological Chemistry | volume = 281 | issue = 16 | pages = 10691–7 | date = April 2006 | pmid = 16481328 | doi = 10.1074/jbc.M511373200 | doi-access = free }}</ref>
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== References ==
{{Reflist|32em}}

== External links ==
* {{MeshName|Ribonucleotide+reductases}}
* [http://rnrdb.molbio.su.se The ribonucleotide reductase database (RNRdb)] {{Webarchive|url=https://web.archive.org/web/20181015063012/http://rnrdb.molbio.su.se/ |date=2018-10-15 }}

{{Nucleotide metabolism}}
{{Other oxidoreductases}}
{{Enzymes}}
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[[Category:EC 1.17.4]]

[[it:Ribonucleoside-trifosfato reduttasi]]