Publications

• Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes.

  Munier-Lehmann H., Chaffotte A., Pochet S., Labesse G.

  We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denatura ... >> More

  We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3'-azido-3'-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKMT: We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKMT: This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials. << Less

  Protein Sci. 10:1195-1205(2001) [PubMed] [EuropePMC]

• Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity.

  Daws T.D., Fuchs J.A.

  Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagen ... >> More

  Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map. << Less

  J Bacteriol 157:440-444(1984) [PubMed] [EuropePMC]