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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.
AuthorsEduard Torrents, Margareta Sahlin, Daniele Biglino, Astrid Gräslund, Britt-Marie Sjöberg
JournalProceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 102 Issue 50 Pg. 17946-51 (Dec 13 2005) ISSN: 0027-8424 [Print] United States
PMID16322104 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Bacterial Proteins
  • Free Radical Scavengers
  • Hydroxylamines
  • Tyrosine
  • NrdF protein, bacteria
  • Ribonucleotide Reductases
  • Hydroxyurea
Topics
  • Amino Acid Sequence
  • Bacillus anthracis (drug effects, genetics, growth & development)
  • Bacterial Proteins (chemistry, genetics, metabolism)
  • Base Sequence
  • Cell Proliferation (drug effects)
  • Cloning, Molecular
  • Cluster Analysis
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli
  • Free Radical Scavengers (pharmacology)
  • Hydroxylamines (pharmacology)
  • Hydroxyurea (pharmacology)
  • Molecular Sequence Data
  • Ribonucleotide Reductases (chemistry, genetics, metabolism)
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Spectrum Analysis
  • Survival Analysis
  • Tyrosine (chemistry, genetics, metabolism)

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