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Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase

¹ Department of Anatomy, Physiology and Biochemistry, The Swedish University of Agricultural Sciences, The Biomedical Centre, S-751 23 Uppsala, Sweden
² Laboratoire d'Enzymologie Moléculaire et Fonctionelle, FRE 2852-CNRS-Université, Paris 6, 75005 Paris, France

(RECEIVED December 21, 2007; FINAL REVISION May 23, 2008; ACCEPTED May 28, 2008)

Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K _m and V _max values of 33 µM and 48 µmol mg^–1 min^–1, respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and D-FMAU was a strong inhibitor with an IC₅₀ value of 10 µM, while other nucleosides—L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine—were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.

Keywords: Bacillus anthracis; thymidylate kinase; TMPK; structure model; nucleoside analogs; drug design; FMAU

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