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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 degrees rotational angle of C(alpha)-(C(beta)-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.

SUBMITTER: Torrents E

PROVIDER: S-EPMC1312384 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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

Torrents Eduard E Sahlin Margareta M Biglino Daniele D Gräslund Astrid A Sjöberg Britt-Marie BM

Proceedings of the National Academy of Sciences of the United States of America 20051201 50

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 I ...[more]

PMID: 16322104

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Project description: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 g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-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, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-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 g(1)-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.

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

Publications

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

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