Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes.
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.
Munier-Lehmann
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Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France. hmunier@pasteur.fr
Chaffotte
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Pochet
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Labesse
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D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry*; D010766:Phosphorylation; D010957:Plasmids / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
D011993:Recombinant Fusion Proteins; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase