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.

The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. The "activase" contains iron, required for activity. The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase. For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.

It has been recently recognized that the class III anaerobic ribonucleotide reductase requires the presence of a second activating gene product, NrdG. We have proposed that the role for NrdG involves the generation of an oxygen sensitive glycyl free radical within the NrdD enzyme. In this article we present the generation of such a glycyl free radical within the T4 NrdD subunit and its dependence upon the phage NrdG subunit. Initially, an overexpression system was created that allowed the joint production of T4 NrdD and T4 NrdG. With this system and in the presence of T4 NrdG, an oxygen-sensitive cleavage of NrdD was observed that mimicked the cleavage observed in phage infected Escherichia coli extracts. Under anaerobic conditions the presence of T4 NrdD with NrdG revealed a strong doublet EPR signal (g ‫؍‬ 2.0039). Isotope labeling of the NrdD with [ H]glycine and [13 C]glycine, respectively, confirmed the presence of a stabilized glycine radical. The unpaired electron is strongly coupled to C-2 in glycine and the doublet splitting originates from one of the ␣-protons. The glycine residue at position 580 was determined to be the radical containing residue through sitedirected mutagenesis studies involving a G580A NrdD mutant. The glycyl radical generation was specific for the T4 NrdG, and the host E. coli NrdG was found to be unable to activate the phage reductase. Finally, anaerobic purification revealed the holoenzyme complex to contain iron, whereas the NrdD polypeptide was found to lack the metal. Our results suggest a tetrameric structure for the T4 anaerobic ribonucleotide reductase containing one homodimer each of NrdD and NrdG, with a single glycyl radical present.

The subtle differences in base size and hydrogen bonding pattern at the effector site are communicated to major conformational changes in the active site. We propose that the altered overlap of Phe-194 with the substrate base governs hydrogen bonding patterns with main and side chain hydrogen bonding groups in the active site. The relevance for evolution is discussed.

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX 2 C-CX 2 C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.

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. Ribonucleotide reductase (RNR)1 catalyzes the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides in all organisms. This is the only de novo way for the cell to make use of the deoxyribonucleotides as building blocks in DNA synthesis (1). Three classes of RNRs are known that differ in quaternary structure and cofactor requirement. Despite these differences, they all catalyze the reduction of ribonucleotides by a related radical-based mechanism (2).All RNRs, except those of herpesviruses (3, 4), are allosterically regulated by deoxyribonucleoside triphosphates and ATP, such that DNA precursors are supplied in pools balanced according to the base composition of the different genomes (5, 6). The allosteric regulation of Escherichia coli class Ia RNR has been meticulously characterized by enzyme activity measurements, nucleotide binding, photoaffinity labeling, affinity chromatography, site-directed mutagenesis, and structure determinations (7-14). The net result of these findings is that ATP and dATP are positive effectors for reduction of pyrimidine ribonucleotides, whereas dTTP stimulates reduction of guanine ribonucleotides, and dGTP stimulates reduction of adenine ribonucleotides. Apart from this allosteric regulation, which is controlled at the so-called specificity site, class Ia RNRs contain another allosteric site denoted the overall activity site, which binds only ATP or dATP. ATP acts (directly or indirectly) as a posit...

Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycyl radical to catalyze the reduction of ribonucleotides to deoxyribonucleotides and formate as ultimate reductant. The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cys-79 and Cys-290 in bacteriophage T4 NrdD numbering. Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction. The recently solved three-dimensional structure of class III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two highly conserved asparagines, Asn-78 and Asn-311, are positioned close to the essential Cys-79. We have investigated the function of Asn-78 and Asn-311 by sitedirected mutagenesis and measured enzyme activity and glycyl radical formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A/N311A) mutant proteins. Our results suggest that both asparagines are important for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn 3 Ala mutant proteins. A plausible role for these two asparagines could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79. This would also control the highly reactive carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide. A detailed reaction scheme including the function of the two asparagines and two formate molecules is proposed for class III RNRs.The building blocks for DNA synthesis, the four deoxyribonucleotides are obtained de novo by reduction of ribonucleotides. Ribonucleotide reductase (RNR) 1 is the enzyme that catalyzes this reaction in all organisms. This seemingly simple reaction, the exchange of the 2Ј-hydroxyl group for a hydrogen atom, requires complex free radical chemistry.Currently, there are three known classes of RNR with a common structural fold (1-3) but little overall sequence similarity (4 -7). The classification of the RNRs is mainly based on the different systems developed by these enzymes to generate a transient thiyl radical, the formation of which initiates the reaction with the ribonucleotide substrate. The aerobic class I enzymes generate within one subunit (R2) a stable tyrosyl radical that via a long-range radical transfer pathway is delivered at the active site cysteine located in the other subunit (R1). The class II enzymes, indifferent to oxygen, use adenosylcobalamin as a thiyl radical generator. The focus of this study, the class III enzymes use a stable glycyl radical within the NrdD protein (the reductase) as a thiyl radical generator. The glycyl radical is formed by homolytic cleavage of the cofactor S-adenosylmethionine (AdoMet), a reaction catalyzed by the oxidation of an ϩ -center in the activase protein (NrdG) (8). The RNR class III activation mechanism, i.e. the AdoMet cle...

We have used 8-azidoadenosine 5-triphosphate (8-N 3 ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage. Saturation studies revealed two saturable sites for this photoaffinity analog of ATP. One site exhibited half-maximal saturation at approximately 5 M [␥-32 P]8-N 3 ATP, whereas the other site required 45 M. To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al 3؉ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses. The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis. Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289 -291 and the other site to the region corresponding to residues 147-160. Site-directed mutagenesis of Cys 290 , a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity. These observations indicate that Cys 290 may reside in or near the active site. High performance liquid chromatography analysis revealed that photoinsertion of [␥-32 P]8-N 3 ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 M dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 M ATP, GTP, CTP, or dCTP had virtually no effect. Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases. There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli.Ribonucleotide reductases catalyze the reduction of ribonucleotides to their corresponding 2Ј-deoxyribonucleotides. Currently, they are divided into three classes based on differences in cofactor requirements, structural composition, and type of radical employed for catalysis (1, 2). Because of the importance of maintaining a balanced supply of deoxyribonucleotides for DNA synthesis (3, 4), they are enzymes that are subject to complex allosteric regulation (5, 6). Although there may be striking differences in primary sequence, the same general mechanism for allosteric regulation appears to apply to all ribonucleotide reductases described to date, with only subtle differences observed (7). Three different nucleotide-binding sites have been localized on the prototypical class I reductase from Escherichia coli using photoaffinity labeling (8) and x-ray crystallography (9, 10). Two of these sites are allosteric sites that coordinate the reduction of all four ribonucleotide substrates at a single active site. One allosteric site binds only ATP and dATP and regulates the overall activity of the enzyme. The other allosteric site, via i...