Mechanisms of Substrate Selectivity for Bacillus Anthracis Thymidylate Kinase

Protein Sci. 2008 Sep;17(9):1486-93. doi: 10.1110/ps.034199.107. Epub 2008 Jun 3.

Abstract

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 microM and 48 micromol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was approximately 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 IC(50) value of 10 microM, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Antineoplastic Agents / pharmacology
  • Antiviral Agents / pharmacology
  • Bacillus anthracis / enzymology*
  • Catalysis
  • Computational Biology
  • Deoxyadenine Nucleotides / metabolism
  • Dimerization
  • Dose-Response Relationship, Drug
  • Drug Design
  • Histidine / metabolism
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Models, Molecular
  • Molecular Weight
  • Phosphorylation
  • Protein Conformation
  • Pyrimidine Nucleosides / pharmacology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Stereoisomerism
  • Substrate Specificity
  • Thymidine Kinase / chemistry
  • Thymidine Kinase / genetics
  • Thymidine Kinase / metabolism*
  • Thymidine Monophosphate / analogs & derivatives
  • Thymidine Monophosphate / antagonists & inhibitors
  • Thymidine Monophosphate / metabolism

Substances

  • Antineoplastic Agents
  • Antiviral Agents
  • Deoxyadenine Nucleotides
  • Pyrimidine Nucleosides
  • Recombinant Proteins
  • Thymidine Monophosphate
  • Histidine
  • Adenosine Triphosphate
  • Thymidine Kinase
  • 2'-deoxyadenosine triphosphate