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 (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...

Background: Ribonucleotide reductase (RNR) makes DNA building blocks. Results: Binding of three dATP molecules to the Pseudomonas aeruginosa class I RNR ␣ subunit inactivates the enzyme by inducing an inert ␣ 4 complex. Conclusion: The number of bound dATP molecules and the tetrameric complex are unique among RNRs. Significance: The novel inhibition mechanism of P. aeruginosa RNR is a potential drug target.

Background: Class Ib ribonucleotide reductase of the severe pathogen Bacillus anthracis can be loaded with manganese or iron.Results: The manganese form was 10-fold more active than the iron form in the presence of the physiological protein NrdH-redoxin.Conclusion: The manganese form is important in the life cycle of B. anthracis.Significance: The physiologically relevant form of ribonucleotide reductase controls B. anthracis proliferation and survival.

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.