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.     

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.     

Ribonucleotide reductases: the link between an RNA and a DNA world?

The structures of a class III ribonucleotide reductase (RNR) and pyruvate formate lyase exhibit striking homology within their active site domains with respect to each other and to the previously published structure of a class I RNR. The common structures and the common complex-radical-based chemistry of these systems, as well as of the class II RNRs, suggest that RNRs evolved by divergent evolution and provide an essential link between the RNA and DNA world.     

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 reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

Ribonucleotide reduction - horizontal transfer of a required function spans all three domains

Background  

Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes.     

Structure and function of the radical enzyme ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3′-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.     

Structure, function, and mechanism of ribonucleotide reductases

Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2'-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR. This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR. Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an alpha/beta-barrel structural motif. This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR. The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase. Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins. There are also significant variations within each of the three classes of RNR. With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli. The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms. In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity. A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR. Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle.     

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, β, 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.     

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.     

Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.     

Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

Ribonucleotide reductases (RNRs) are used by all free‐living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical‐based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo‐velocity and sequence similarity network (SSN) analyses, we show that the N‐terminal regulatory motif known as the ATP‐cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C‐terminal extensions of class II RNRs can contain folded domains that share homology with an Fe‐S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.     

Novel mutator mutants of E. coli nrdAB ribonucleotide reductase: insight into allosteric regulation and control of mutation rates

Ribonucleotide reductase (RNR) is the enzyme critically responsible for the production of the 5'-deoxynucleoside-triphosphates (dNTPs), the direct precursors for DNA synthesis. The dNTP levels are tightly controlled to permit high efficiency and fidelity of DNA synthesis. Much of this control occurs at the level of the RNR by feedback processes, but a detailed understanding of these mechanisms is still lacking. Using a genetic approach in the bacterium Escherichia coli, a paradigm for the class Ia RNRs, we isolated 23 novel RNR mutants displaying elevated mutation rates along with altered dNTP levels. The responsible amino-acid substitutions in RNR reside in three different regions: (i) the (d)ATP-binding activity domain, (ii) a novel region in the small subunit adjacent to the activity domain, and (iii) the dNTP-binding specificity site, several of which are associated with different dNTP pool alterations and different mutational outcomes. These mutants provide new insight into the precise mechanisms by which RNR is regulated and how dNTP pool disturbances resulting from defects in RNR can lead to increased mutation.