Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

Ribonucleotide reductase from Escherichia coli consists of two dissociable, nonidentical homodimeric proteins called R1 and R2. The role of the C-terminal region of R2 in forming the R1R2 active complex has been studied. A heterodimeric R2 form with a full-length polypeptide chain and a truncated one missing the last 30 carboxyl-terminal residues was engineered by site-directed mutagenesis. Kinetic analysis of the binding of this protein to R1, compared with full-length or truncated homodimer, revealed that the C-terminal end of R2 accounts for all of its interactions with R1. The intrinsic dissociation constant of the heterodimeric R2 form, with only one contact to R1, 13 microM, is of the same magnitude as that obtained previously [Climent, I., Sjöberg, B.-M.,& Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18 microM. We have also mutagenized the only two invariant residues localized at the C-terminal region of R2, glutamic acid-350 and tyrosine-356, to alanine. The binding of these mutant proteins to R1 remains tight, but their catalytic activity is severely affected. While E350A protein exhibits a low (240 times less active than the wild-type) but definitive activity, Y356A is completely inactive. A catalytic rather than structural role for these residues is discussed.

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.

Two different tryptophan radicals (Wa • and Wb •) with lifetimes of several minutes at room temperature are formed during the reconstitution of the diiron center in the Escherichia coli ribonucleotide reductase mutant protein R2 Y122F. Detailed hyperfine parameters are for the first time determined for protein-linked oxidized neutral tryptophan radicals. Wa • is freeze-trapped and investigated by EPR and ENDOR in protonated and selectively deuterated proteins at 20 K. Two hyperfine couplings from the β-methylene protons, hyperfine tensors of two α-protons, and the complete nitrogen hyperfine tensor are determined. Based on the absence of a large hyperfine coupling from the N−H proton, which would be expected for a cation radical, and on comparison of the experimental data with theoretical spin densities from density functional calculations, Wa • is assigned to an oxidized neutral tryptophan radical. A small anisotropic hyperfine coupling detected in selectively deuterated Wa • is tentatively assigned to a proton which is hydrogen bonded to the nitrogen of Wa •. A similar spin density distribution as for Wa • is obtained also for the second tryptophan radical, Wb •, observed by EPR at room temperature, which is also assigned to an oxidized neutral radical.

BackgroundRibonucleotide 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.DescriptionIn 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.ConclusionRNRdb 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.

The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by sitedirected mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2 -azido-2 -deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDPderived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogenbonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.The enzyme ribonucleotide reductase is essential for all living organisms. By catalyzing the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the enzyme furnishes cells with precursors for DNA synthesis. To maintain a stable and balanced supply of nucleotides during cell proliferation, the enzyme is cell cycle regulated (1) and also under strict allosteric regulation (for recent review see Ref.2). The Escherichia coli ribonucleotide reductase holoenzyme complex consists of two nonidentical dimeric proteins, R1 and R2, which are each inactive alone. The larger protein R1 contains substrate and allosteric effector binding sites. The substrate binding site of R1 includes a cysteine triad that is involved in catalysis (3-5). The smaller protein R2 contains an essential tyrosyl radical that is generated and stabilized by an oxo-bridged diiron center (6 -9). The oxidized tyrosyl radical of R2 probably participates in the reaction mechanism as a transient electron sink.The reaction catalyzed by ribonucleotide reductase is the reduction of the 2Ј-hydroxyl group of a ribonucleoside diphosphate. The mechanism involves the initial generation of a transient protein radical in R1 close to the bound substrate. The protein radical abstracts a hydrogen atom from the 3Ј position of the substrate thereby generating an oxidized substrate radical that enables leaving the protonated 2Ј-hydroxyl group. The resulting substrate radical cation intermediate is reduced by a redox-active cysteine pair, which in turn is oxidized to a disulfide (10). The 3Ј-hydrogen atom is reintroduced by the same amino acid that initially abstracted it and that again forms the transient protein radical (11).The crystal structure of the R2 protein shows that the R2 radical is buried inside the protein structure about 10 Å from the closest s...

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (␣) and R2 (␤). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited ␣ 4 ␤ 4 complex in the presence of dATP and an active ␣ 2 ␤ 2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30 -50 times stronger in the ␣ 4 ␤ 4 complex than in the ␣ 2 ␤ 2 complex, which was in equilibrium with free ␣ 2 and ␤ 2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form ␣ 4 ␤ 4 complexes. ATP could also induce the formation of a generally inhibited ␣ 4 ␤ 4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for ␣ 4 ␤ 4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/ dTTP and forms active and inactive ␣ 6 ␤ 2 complexes. Ribonucleotide reductase (RNR)3 is a key enzyme for the synthesis of DNA building blocks as it converts ribonucleoside-5Ј-dior triphosphates to the corresponding deoxyribonucleotides (1). It is important that the synthesis of these building blocks is regulated to avoid an increased mutation rate caused by perturbed dNTP levels (2, 3). RNRs can be divided into three different classes (I, II, and III) mainly based on different cofactors for the catalytic activity, oxygen dependence, and mechanism for free radical generation (1). Class Ia RNRs (1) contain two non-identical subunits, denoted R1 (␣) and R2 (␤), that are both needed for enzyme activity. In bacteria, the two subunits are often referred to as NrdA and NrdB, respectively. The required radical for ribonucleotide reduction is generated on a tyrosine residue in the R2 protein and transferred via a radical transfer pathway to the R1 protein where the catalysis occurs. The R1 protein contains the substrate binding site and two allosteric sites termed the specificity and overall activity sites. Based on differences in allosteric regulation and polypeptide sequence, class I RNRs are further subgrouped into class Ia and Ib (4). Class Ia enzymes have an overall activity site, whereas class Ib lacks the N-terminal region where the overall activity site is located.Class Ia RNRs are found almost in all eukaryotic organisms and some bacteria, viruses, and bacteriophages (see the Ribonucleotide Reductase Database (RNRdb)). The first RNR studied was from...

Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.