Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity

DNA building blocks: keeping control of manufacture

Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: α2 and β2. The α subunit contains the site of nucleotide reduction, and the β subunit contains the essential diferric-tyrosyl radical (Y•) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (α) and NrdB (β), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (α) and NrdF (β), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y• levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y• maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class Ib RNR. It possesses redox properties unprecedented for a flavodoxin (Eox/sq = −264 ± 17 mV and Esq/hq = −255 ± 17 mV) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE

Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical

Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60° rotational angle of C(α)-(C(β)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens

The Dimanganese(II) Site of Bacillus subtilis Class Ib Ribonucleotide Reductase

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn[III over 2]-Y[superscript •], in their homodimeric NrdF (β2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn[II over 2] form of NrdF is an important component in understanding O[subscript 2]-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn[II over 2]-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 Å resolution crystal structure of Bacillus subtilis Mn[II over 2]-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the β2 dimer are distinct from those observed in E. coli Mn[II over 2]-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn[II over 2] cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn[III over 2]-Y[superscript •] cofactor assembly in class Ib RNRs.United States. Dept. of Energy (Contract DE-AC02-06CH11357)Michigan Economic Development Corporation (Michigan Technology Tri-Corridor Grant 085P1000817)National Cancer Institute (U.S.) (Y1-CO-1020)National Institute of General Medical Sciences (U.S.) (Y1-GM-1104

Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase

Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α2) and the radical-generation subunit (β2) interact. Here we present the first structure of a complex between α2 and β2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α4β4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α2β2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α2β2 and α4β4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α2β2 and α4β4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli