Class I ribonucleotide reductases: metallocofactor assembly and repair in vitro and in vivo.

Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.

The periodic table of ribonucleotide reductases.

In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2'-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, ß, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.

A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site.

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor.

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

Generation of a stable, aminotyrosyl radical-induced α2ß2 complex of Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (ß2) subunit over a 35-Å pathway of redox-active amino acids: Y122• ↔ [W48?] ↔ Y356 in ß2 to Y731 ↔ Y730 ↔ C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730• is generated in α2 in the presence of wild-type (wt)-ß2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730• induces formation of a kinetically stable α2ß2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-ß2 is 25-fold tighter (Kd = 7 nM) than for wt-α2

The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y·) or a dimanganese-Y· cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe 2 (III) -Y· cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe 2 (III) -Y· cofactor. We also report enzymatic activity of Fe 2 (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.

Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer.

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ~35 Å from the stable diferric/tyrosyl-radical (Y122(•)) cofactor in the ß subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the ß sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122(•) by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122(•), but also directly in catalysis.

Ribonucleotide reductase as a target to control apicomplexan diseases.

Malaria is caused by species in the apicomplexan genus Plasmodium, which infect hundreds of millions of people each year and kill close to one million. While malaria is the most notorious of the apicomplexan-caused diseases, other members of eukaryotic phylum Apicomplexa are responsible for additional, albeit less well-known, diseases in humans, economically important livestock, and a variety of other vertebrates. Diseases such as babesiosis (hemolytic anemia), theileriosis and East Coast Fever, cryptosporidiosis, and toxoplasmosis are caused by the apicomplexans Babesia, Theileria, Cryptosporidium and Toxoplasma, respectively. In addition to the loss of human life, these diseases are responsible for losses of billions of dollars annually. Hence, the research into new drug targets remains a high priority. Ribonucleotide reductase (RNR) is an essential enzyme found in all domains of life. It is the only means by which de novo synthesis of deoxyribonucleotides occurs, without which DNA replication and repair cannot proceed. RNR has long been the target of antiviral, antibacterial and anti-cancer therapeutics. Herein, we review the chemotherapeutic methods used to inhibit RNR, with particular emphasis on the role of RNR inhibition in Apicomplexa, and in light of the novel RNR R2_e2 subunit recently identified in apicomplexan parasites.

Implications of the inability of Listeria monocytogenes EGD-e to grow anaerobically due to a deletion in the class III NrdD ribonucleotide reductase for its use as a model laboratory strain.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that causes life-threatening diseases in humans. It grows and survives in environments of low oxygen tension and under conditions of strict anaerobiosis. Oxygen-limiting conditions may be an important factor in determining its pathogenicity. L. monocytogenes serovar 1/2a strain EGD-e has been employed intensively to elucidate the mechanisms of intracellular multiplication and virulence. Listeria possesses genes encoding class I aerobic and class III anaerobic ribonucleotide reductases (RNRs). The class III RNR consists of a catalytic subunit NrdD and an activase NrdG. Surprisingly, L. monocytogenes EGD-e, but not other L. monocytogenes strains or other listerial species, is unable to grow under strict anaerobic conditions. Inspection of listerial NrdD amino acid sequences revealed a six-amino acid deletion in the C-terminal portion of the EGD-e protein, next to the essential glycyl radical domain. Nevertheless, L. monocytogenes EGD-e can grow under microaerophilic conditions due to the recruitment of residual class Ia RNR activity. A three-dimensional (3D) model based on the structure of bacteriophage T4 NrdD identified the location of the deletion, which appears in a highly conserved part of the NrdD RNR structure, in the α/ß barrel domain near the glycyl radical domain. The deleted KITPFE region is essential either for interactions with the NrdG activase or, indirectly, for the stability of the glycyl radical loop. Given that L. monocytogenes EGD-e lacks a functional anaerobic RNR, the present findings are relevant to the interpretation of studies of pathogenesis with this strain specifically, in particular under conditions of low oxygen tension.

Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.

Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and ß (NrdF). ß contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)(2)-Y(•)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/ß(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(•) [Mn(III)(2)-Y(•)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(•) and Fe(III)(2)-Y(•) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(•)/ß(2)). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)(2)-Y(•) center with 2 Mn/ß(2) and 0.5 Y(•)/ß(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(•)/ß(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(•) enzyme.

An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase.

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: alpha2 (NrdE), where nucleotide reduction occurs, and beta2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)(6)-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn(II) and incubated with fully reduced NrdI (NrdI(hq)) and O(2). Active RNR was rapidly produced with 0.25 +/- 0.03 tyrosyl radical (Y*) per beta2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is Mn(III)(2)-Y*, which we propose is generated by Mn(II)(2)-NrdF reacting with two equivalents of HO(2)(-), produced by reduction of O(2) by NrdF-bound NrdI(hq). In the absence of NrdI(hq), with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe(II) and incubated with O(2) in the presence or absence of NrdI(hq) gave 0.2 and 0.7 Y*/beta2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI(hq) hinders Fe(III)(2)-Y* cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn(III)(2)-Y* cofactor, not the diferric-Y* one, is the active metallocofactor in vivo.

Ribonucleotide reductases: substrate specificity by allostery.

Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.

RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank.

BACKGROUND: Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes. DESCRIPTION: In RNRdb (Ribonucleotide Reductase database), we have collated and curated all known RNR protein sequences with the aim of providing a resource for exploration of RNR diversity and distribution. By comparing expert manual annotations with annotations stored in Genbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23% of protein sequences included in RNRdb are correctly annotated across the key attributes of class, role and function, with 17% being incorrectly annotated across all three categories. This illustrates the utility of specialist databases for applications where a high degree of annotation accuracy may be important. The database houses information on annotation, distribution and diversity of RNRs, and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb is accessible through a public web interface at http://rnrdb.molbio.su.se. CONCLUSION: RNRdb is a specialist database that provides a reliable annotation and classification resource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. The recent expansion in available genome sequence data have provided us with a picture of RNR distribution that is more complex than believed only a few years ago; our database indicates that RNRs of all three classes are found across all three cellular domains. Moreover, we find a number of organisms that encode all three classes.

Hierarchical classification of functionally equivalent genes in prokaryotes.

Functional classification of genes represents a fundamental problem to many biological studies. Most of the existing classification schemes are based on the concepts of homology and orthology, which were originally introduced to study gene evolution but might not be the most appropriate for gene function prediction, particularly at high resolution level. We have recently developed a scheme for hierarchical classification of genes (HCGs) in prokaryotes. In the HCG scheme, the functional equivalence relationships among genes are first assessed through a careful application of both sequence similarity and genomic neighborhood information; and genes are then classified into a hierarchical structure of clusters, where genes in each cluster are functionally equivalent at some resolution level, and the level of resolution goes higher as the clusters become increasingly smaller traveling down the hierarchy. The HCG scheme is validated through comparisons with the taxonomy of the prokaryotic genomes, Clusters of Orthologous Groups (COGs) of genes and the Pfam system. We have applied the HCG scheme to 224 complete prokaryotic genomes, and constructed a HCG database consisting of a forest of 5339 multi-level and 15 770 single-level trees of gene clusters covering approximately 93% of the genes of these 224 genomes. The validation results indicate that the HCG scheme not only captures the key features of the existing classification schemes but also provides a much richer organization of genes which can be used for functional prediction of genes at higher resolution and to help reveal evolutionary trace of the genes.

The evolution of ribonucleotide reduction revisited.

Ribonucleotide reductases (RNRs) catalyze the conversion of both purine and pyrimidine nucleotides to deoxynucleotides in all organisms and provide all the monomeric precursors essential for both DNA replication and repair. RNRs have been divided into three classes on the basis of their unique metallo-cofactors. The exquisitely controlled free radical chemistry used by all RNRs, and the commonality of the structures of the subunits where the nucleotide reduction process occurs, together provide compelling evidence for the importance of chemistry in the divergent evolution of RNRs from a common progenitor.

The evolution of ribonucleotide reduction.

Ribonucleotide reduction was essential for the transition from RNA to DNA by supplying deoxyribonucleotide precursors. The reaction requires free radical chemistry. Three quite different classes of ribonucleotide reductases are known today. All three are proteins containing a stable free radical amino acid, but each uses a different mechanism for its generation. Did they evolve from a common ancestor, with the arrival of atmospheric oxygen providing the driving force for their divergence, or was each a separate evolutionary invention?

Circular dichroism and magnetic circular dichroism studies of the biferrous site of the class Ib ribonucleotide reductase from Bacillus cereus: comparison to the class Ia enzymes.

The rate limiting step in DNA biosynthesis is the reduction of ribonucleotides to form the corresponding deoxyribonucleotides. This reaction is catalyzed by ribonucleotide reductases (RNRs) and is an attractive target against rapidly proliferating pathogens. Class I RNRs are binuclear non-heme iron enzymes and can be further divided into subclasses. Class Ia is found in many organisms, including humans, while class Ib has only been found in bacteria, notably some pathogens. Both Bacillus anthracis and Bacillus cereus encode class Ib RNRs with over 98% sequence identity. The geometric and electronic structure of the B. cereus diiron containing subunit (R2F) has been characterized by a combination of circular dichroism, magnetic circular dichroism (MCD) and variable temperature variable field MCD and is compared to class Ia RNRs. While crystallography has given several possible descriptions for the class Ib RNR biferrous site, the spectroscopically defined active site contains a 4-coordinate and a 5-coordinate Fe(II), weakly antiferromagnetically coupled via mu-1,3-carboxylate bridges. Class Ia biferrous sites are also antiferromagnetically coupled 4-coordinate and 5-coordinate Fe(II), however quantitatively differ from class Ib in bridging carboxylate conformation and tyrosine radical positioning relative to the diiron site. Additionally, the iron binding affinity in B. cereus RNR R2F is greater than class Ia RNR and provides the pathogen with a competitive advantage relative to host in physiological, iron-limited environments. These structural differences have potential for the development of selective drugs.

Ribonucleotide reductases: influence of environment on synthesis and activity.

Ribonucleotide reductases (RNRs) are enzymes that provide deoxyribonucleotides (dNTPs), the building blocks required for de novo DNA synthesis and repair. They are found in all organisms from prokaryotes to eukaryotes. Interestingly, in the microbial world, several organisms possess the genes encoding two, or even three different RNRs that present different structures and allosteric regulation. The finding of an increasing number of bacterial species that possess more than one RNR might suggest particular functions for these enzymes in different growth conditions. Recent support for this proposal comes from studies indicating that expression and activity of the different RNRs depends on the environment. The oxygen content as well as the redox and oxidative stresses regulate RNR activity and synthesis in various organisms. This regulation has a direct consequence on dNTP pools. An excess of dNTP pools that leads to misincorporation of dNTPs results in genetic abnormalities in eukaryotes as in prokaryotes. In contrast, increased dNTP concentrations help cells to survive under conditions where DNA has been damaged. Hence the use of different RNRs in response to various environmental conditions allows the cell to regulate the amount precisely of dNTP in both a positive and negative manner so that enough, yet not excessive, dNTPs are synthesized.

Ribonucleotide reductases and their occurrence in microorganisms: a link to the RNA/DNA transition.

The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.