Structural determinants and distribution of phosphate specificity in ribonucleotide reductases

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.     

The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. Here we present the crystal structure of class II (coenzyme B12-dependent) ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the apo enzyme form and in complex with the B12 analog adeninylpentylcobalamin at 1.75 and 2.0 A resolution, respectively. This monomeric, allosterically regulated class II RNR retains all the key structural features associated with the catalytic and regulatory machinery of oligomeric RNRs. Surprisingly, the dimer interface responsible for effector binding in class I RNR is preserved through a single 130-residue insertion in the class II structure. Thus, L. leichmannii RNR is a paradigm for the simplest structural entity capable of ribonucleotide reduction, a reaction linking the RNA and DNA worlds.     

Control of metallation and active cofactor assembly in the class Ia and Ib ribonucleotide reductases: diiron or dimanganese?

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Y?), which can be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y? cofactor. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y? cofactor. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Y? and dimanganese(III)-Y cofactors raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms' growth and virulence.     

Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12-dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.     

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/β 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.     

Starting a new chapter on class Ia ribonucleotide reductases

Ribonucleotide reductases (RNRs) use radical-based chemistry to convert ribonucleotides into deoxyribonucleotides, an essential step in DNA biosynthesis and repair. There are multiple RNR classes, the best studied of which is the class Ia RNR that is found in Escherichia coli, eukaryotes including humans, and many pathogenic and nonpathogenic prokaryotes. This review covers recent advances in our understanding of class Ia RNRs, including a recent reporting of a structure of the active state of the E. coli enzyme and the impacts that the structure has had on spurring research into the mechanism of long-range radical transfer. Additionally, the review considers other recent structural and biochemical research on class Ia RNRs and the potential of that work for the development of anticancer and antibiotic therapeutics.     

Ribonucleotide reductase modularity: Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.