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

Abstract

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 ␣-(C␤-C1)-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.anthrax ͉ electron paramagnetic resonance ͉ hydroxyurea ͉ N-methyl hydroxylamine ͉ bacterial growth inhibition

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…The interaction of S. sanguinis NrdF with its NrdI is also of interest because they belong to a third phylogenetic subgroup that remained uncharacterized until our studies (24,28,51). This subclassification based on bioinformatics is supported by the measured K d of 2.9 M for S. sanguinis NrdI-NrdF interactions, distinct from tighter interactions measured for E. coli (Ͻ0.05 M) and B. subtilis (0.6 M) systems (16).…”

“…This organism can thus also serve as a model for many pathogenic organisms with class Ib RNR as the only aerobic source of deoxynucleotides (27). Fourth, based on phylogenetic analysis (28) 3 H]CDP was obtained from ViTrax and diluted with CDP in buffer A to 6,000 -12,000 cpm/nmol before use. Radioactive samples were analyzed using a Beckman Coulter LS6500 scintillation counter.…”

Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate

“…As discussed above we have previously shown that only the manganese reconstituted form of B. anthracis NrdF shows a substantial enzymatic activity with the physiologically relevant reductants TrxA and NrdH in enzymatic assays (12,13). Additionally, it has been demonstrated that inhibiting B. anthracis RNR using N-methylhydroxylamine efficiently knocks out growth of the organism, making it an attractive target for novel antibiotics (16 (12,15). The FMN content in purified NrdI was determined via acid treatment of the protein to release the FMN cofactor, in accordance with the method of Mayhew and Massey (17).…”

“…Preparation and Characterization of Mn-reconstituted B. anthracis NrdF-The apo-NrdF protein was purified via a modified literature procedure (16), by adding an incubation with 5 mM EDTA under reducing conditions for 2-3 h after which time adventitiously bound metal ions were removed by sizeexclusion chromatography. Finally, apo-NrdF was isolated in Ͼ95% purity by anionic exchange chromatography.…”

“…The apo-form of B. anthracis NrdF was purified as described previously (16) with the following modification: the intermediate purification product obtained following phenyl-Sepharose chromatography was concentrated and the solution was treated with 2.5 mM dithionite and 5 mM EDTA under strictly anaerobic conditions at 4°C for 2-3 h. Following this treatment the resulting reaction mixture was purified by size exclusion chromatography on a Superose column, eluting with 50 mM TrisHCl buffer, pH 7.6, with 100 mM NaCl. Finally, apo-NrdF was loaded onto a Q-Sepharose column and the column was washed with three column volumes of Tris-HCl buffer before apo-NrdF was eluted using a linear gradient from 0 to 500 mM KCl.…”