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

doi:10.1073/pnas.0506410102

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

Departments of ^*Molecular Biology and Functional Genomics and ^†Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved October 13, 2005 (received for review July 27, 2005)

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_α-(C_β-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.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: britt-marie.sjoberg{at}molbio.su.se.
↵ ‡ Present address: Max Planck Institute for Bioinorganic Chemistry, D-45470 Mülheim an der Ruhr, Germany.
Author contributions: E.T., M.S., D.B., A.G., and B.-M.S. designed research, performed research, analyzed data, and wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: DTT, dithiothreitol; HA, hydroxylamine; HU, hydroxyurea; M-HA, N-methyl hydroxylamine; RNR, ribonucleotide reductase.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AJ704809).