A glycyl radical site in the crystal structure of a class III ribonucleotide reductase.

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 ribonucleotide reductase jigsaw puzzle: a large piece falls into place.

The three-dimensional structure of ribonucleotide reductase protein R1 from Escherichia coli reveals a novel 10-stranded alpha/beta barrel fold. A long loop penetrates the center cavity to assemble the active site cysteine triad.

Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases.

BACKGROUND: The specificity of ribonucleotide reductases (RNRs) toward their four substrates is governed by the binding of deoxyribonucleoside triphosphates (dNTPs) to the allosteric specificity site. Similar patterns in the kinetics of allosteric regulation have been a strong argument for a common evolutionary origin of the three otherwise widely divergent RNR classes. Recent structural information settled the case for divergent evolution; however, the structural basis for transmission of the allosteric signal is currently poorly understood. A comparative study of the conformational effects of the binding of different effectors has not yet been possible; in addition, only one RNR class has been studied. RESULTS: Our presentation of the structures of a class III anaerobic RNR in complex with four dNTPs allows a full comparison of the protein conformations. Discrimination among the effectors is achieved by two side chains, Gln-114 and Glu-181, from separate monomers. Large conformational changes in the active site (loop 2), in particular Phe-194, are induced by effector binding. The conformational differences observed in the protein when the purine effectors are compared with the pyrimidine effectors are large, while the differences observed within the purine group itself are more subtle. CONCLUSIONS: 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.

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

New crystal forms of the small subunit of ribonucleotide reductase from Escherichia coli.

The small subunit of ribonucleotide reductase from Escherichia coli has been crystallized in two new crystal forms. The form most suitable for X-ray analysis belongs to the orthorhombic space group P2(1)2(1)2(1). It has the cell dimensions 74.3 A, 85.5 A, 115.7 A and diffracts to about 2.1 A resolution. The asymmetric unit most probably contains one dimer. Absorption spectra of single crystals confirm that the crystals contain a binuclear iron center. Crystals of the iron-depleted apoenzyme have also been obtained.

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison.

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.

The tyrosyl free radical in ribonucleotide reductase.

The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical.

Perception of risk and subjective health among victims of the Chernobyl disaster.

Several studies have demonstrated that the nuclear power plant accident at Chernobyl in 1986 had a strong impact on the subjective health of the inhabitants in the surrounding regions and that the majority of these health complaints appear to be stress-related. An epidemiological survey among the adult population of the Gomel region in Belarus near Chernobyl showed higher rates of self-reported health problems, psychological distress and medical service use in this region than in a comparable unexposed region. This paper presents an analysis of data on cognitive factors that were collected in this study. The findings support the hypothesis that cognitive variables such as risk perception and sense of control play an important role as mediating factors in the explanation of the observed health differences between the exposed and non-exposed regions. A tentative model is presented to further clarify the role of risk perception in the occurrence of non-specific health complaints after such ecological disasters.

Public reaction to radiation: fear, anxiety, or phobia?

Public fear reactions to ionizing radiation are discussed in a social psychological context. The common use of the terms fear, anxiety, panic, and phobia is related to their clinical meanings, and the authors stress the importance of caution when using certain psychiatric terms for interpreting public reactions to radiation. Differences related to existing knowledge and belief structures, trust, and preferences, create obstacles to effective communication; however, the study of such differences also offers explanations to different reactions and different viewpoints. More information and communication on radiation, clear behavioral guidelines in situations of increased radiation levels, and respect for citizens' concerns about radiation protection would counterbalance lay people's fears and feelings of vulnerability. Such measures may enhance familiarity with radiation, increase perceived personal control in anxiety-creating situations, and develop trust in authorities and their expertise.

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.

The Cys292-->Ala substitution in protein R1 of class I ribonucleotide reductase from Escherichia coli has a global effect on nucleotide binding at the specificity-determining allosteric site.

Ribonucleotide reductase from aerobically grown Escherichia coli is allosterically regulated, both with respect to general activity and substrate specificity. Protein R1, the homodimeric enzyme component which harbours binding sites for allosteric effectors (nucleoside triphosphates) as well as substrates (ribonucleoside diphosphates), has been engineered at Cys292 close to the dimer interaction area. This residue was earlier shown to be specifically photoaffinity labelled with the allosteric nucleotide dTTP. In this study the effect of the Cys292-->Ala substitution is shown to be an overall diminished nucleotide binding at the specificity site reflected in Kd values for dTTP, dGTP and dATP higher by more than one order of magnitude with respect to wild type. The mutant protein's interaction with other protein components of the ribonucleotide reductase system was unaffected by the mutation. These results show that Cys292 in protein R1 of class I ribonucleotide reductase from E. coli is located in the allosteric specificity site.

Effect of hydroxyurea on T4 ribonucleotide reductase.

Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides. The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme. Fifty per cent inhibition occurs at 10 micrometer hydroxyurea. Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates. Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation.

The free radical in ribonucleotide reductase from E. coli.

Protein B2, one of the subunits of ribonucleotide reductase from Escherichia coli, contains a stable free radical. It is characterized by a doublet e.p.r. signal centered around g = 2.0047 and a sharp peak at 410 nm in the optical spectrum. The radical has been assigned to a tyrosyl residue in the protein with its spin density delocalized over the aromatic ring. Protein B2 also contains two antiferromagnetically-coupled high-spin iron(III) atoms, which stabilize the free radical. Protein B1, the other subunit of ribonucleotide reductase, contains two binding sites for substrate molecules, which are the four common ribonucleoside diphosphates. It also contains two classes of allosteric effector-binding sites. ATP and deoxyribonucleoside triphosphates function as effectors. A one-to-one complex of proteins B1 and B2 forms the enzymically-active ribonucleotide reductase. The free radical is, most likely, part of the active site.