The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the Rl and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S.typhimurium nrdE and nrdF genes (RlE and R2F Ribonucleotide reductases catalyze the synthesis of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. At least three separate classes of enzymes are known, each with a distinct protein structure but all requiring a protein radical for catalysis (1). The long-studied aerobic Escherichia coli enzyme is the prototype for class I enzymes, also present in all higher organisms and some other microorganisms. E. coli genes nrdA and nrdB encode the a and 8 polypeptide chains, respectively, that form the Rl (a2) and R2 (132) proteins that constitute the enzyme (2). Each protomer of the Rl dimer (2 x 85.7 kDa) contains one substrate-binding site with redox-active thiols involved in the reduction of the substrate ribonucleoside diphosphate, and two separate types of allosteric sites: one, the activity site, controls the overall activity of the enzyme, with ATP as a positive effector and dATP as a negative effector; the other, the substrate-specificity site, controls the specificity of the enzyme, with ATP and dATP favoring pyrimidine reduction, dTTP favoring GDP reduction, and dGTP favoring ADP reduction (3).The R2 dimer (2 x 43.4 kDa) contains two dinuclear iron centers with associated stable tyrosyl free radicals, located at Tyr-122 of the polypeptide chain (4). The drug hydroxyurea scavenges this radical and thereby inactivates the enzyme. Class II and III enzymes lack the tyrosyl radical. Class II enzymes, with the Lactobacillus leichmanni enzyme as a prototype, employ adenosylcobalamin as a radical generator, whereas class III enzymes use S-adenosylmethionine together with iron for this purpose.Salmonella typhimurium contains an active class I enzyme with amino acid sequences 96.5% and 98.4% identical to the E. coli Rl and R2 proteins, respectively (A.J., unpublished results). Recent genetic evidence involving complementation of nrd mutants of E. coli suggested the presence in S. typhimurium of the genes, nrdE and nrdF, coding for a second class I enzyme (5). These genes are also present on the chromosome of E. coli but are, under standard growth conditions, expressed only when present on a plasmid. The amino acid sequences deduced for the corresponding proteins showed a limited identity with other class I enzymes but contained many of their catalytically important residues.We have purified and characterized the two proteins encoded by the cloned genes nrdE and nrdF from S. typhimurium. Each protein is a homodimer. Together they catalyze the reductio...

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia cofi. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dAl, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map ofE. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dAl is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dAl activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.During anaerobic growth, Escherichia coli induces an enzyme that catalyzes the reduction of CTP to dCTP (7-9, 11). The gene for this enzyme was recently cloned (28) and found to be distinct from nrdA and nrdB, which code for the aerobic ribonucleoside diphosphate reductase (29). In the active state, both enzymes contain organic radicals as part of their protein structures and iron as a cofactor (19,29). In the aerobic enzyme, the radical is located on and the enzyme contains a diferric center with the iron ions linked by a ,u-oxo-bridge (29). A glycyl residue was suggested to harbor the organic radical of the anaerobic reductase, whose iron center consists of an iron-sulfur cluster (19,28).A difference between the two enzymes is that the aerobic reductase, as isolated, shows full activity and contains the radical in stable form, whereas the isolated anaerobic enzyme is inactive and lacks the radical. Instead, the enzyme activity and radical of the latter enzyme appear only after anaerobic incubation of the isolated protein with NADPH, S-adenosylmethionine, and two uncharacterized E. coli fractions, provisionally called dAl and RT (8

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.

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD ؊ mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.Ribonucleotide reductases are essential enzymes that catalyze the reduction of ribonucleoside di-or triphosphates and thereby provide the building blocks required for DNA replication and repair. Three different classes of enzymes are known (1), each with a distinct protein structure but all requiring a protein radical for catalysis and all regulated by similar allosteric effects.Class I reductases are aerobic enzymes present in all higher organisms and certain microorganisms, among them Escherichia coli (2). This bacterium actually has the potential to produce two separate class I enzymes. One of them, coded for by the nrdA and -B genes (3) is the functional enzyme during the growth of E. coli and has been the prototype for all class I enzymes. The second enzyme is coded for by the nrdE and -F genes (4), first discovered in Salmonella typhimurium (5), and is normally not fully functional. Expression of the chromosomal nrdEF genes thus is not sufficient to complement mutations in nrdAB (5). The two enzymes show a limited sequence similarity but contain certain strategical amino acids in identical positions. They differ to some extent in their allosteric regulation and with respect to their hydrogen donors (6). A functionally active reductase of the NrdEF-type was recently found in Mycobacterium tuberculosis (7). Mycoplasma genitalium contains the nrdEF genes but not the nrdAB genes (8).All class I enzymes consist of two proteins that are named R1 and R2 for NrdAB enzymes (2) and R1E and R2F (6) for the NrdEF enzymes. Each protein has specific functions: R1 and R1E contain the binding sites for both substrates and allosteric effectors and carry out the actual reduction of the ribonucleotide. R2 and R2F contain diferric iron centers...

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.Ribonucleotides are reduced to deoxyribonucleotides by ribonucleotide reductases, which are radical containing enzymes that may be divided into three main classes (1, 2).The electrons for this reaction are supplied by small redoxactive proteins such as thioredoxin (Trx) 1 or glutaredoxin (Grx) in the case of class I and II ribonucleotide reductase (3, 4), whereas formate fulfills this function for the anaerobic class III enzyme (5). Thioredoxin and glutaredoxin both contain two redox-active cysteine thiols in their reduced form, which by dithiol-disulfide interchange reduce an acceptor disulfide in the active center of ribonucleotide reductase. The active site sequences of thioredoxins and glutaredoxins are conserved among species, being Cys-Gly-Pro-Cys for thioredoxin and CysPro-Tyr-Cys for glutaredoxin (3, 4). The disulfide in oxidized thioredoxin is regenerated to a dithiol by thioredoxin reductase (TR) and NADPH, whereas oxidized glutaredoxin is reduced by 2 mol of GSH with the formation of GSSG, which is reduced by glutathione reductase (GR) and NADPH. Escherichia coli contains three different glutaredoxins (called Grx1, -2, and -3 (6)), which, like all glutaredoxins from other species, show high activity as general GSH-disulfide oxidoreductases in a coupled system with GSH, NADPH, and glutathione reductase (7). Three-dimensional structures for thioredoxins (8) and glutaredoxins (9, 10) show that they have essentially completely unrelated amino acid sequences but a similar overall fold (often referred to as the thioredoxin fold), con...