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Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase

¹ The Swedish University of Agricultural Biosciences;
² Swedish University of Agricultural Science;
³ Laboratoire d'Enzymologie Moleculaire et FonctionelleFRE 2852-CNRS-Universite Paris

(RECEIVED December 21, 2007; 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 Km and Vmax values of 33 µM and 48 µmol/mg/min, 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: Enzymes; Active sites; Structure/function studies; Molecular weight determination; Kinetics; Enzyme Inhibitors; Bacillus anthracis; FMAU; Thymidylate kinase

⁴ E-mail: staffan.eriksson{at}afb.slu.se

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