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Protein Science (2001), 10:1195-1205.
Copyright © 2001 The Protein Society

Thymidylate kinase of Mycobacterium tuberculosis: A chimera sharing properties common to eukaryotic and bacterial enzymes

HÉLÈNE Munier-Lehmann1, Alain Chaffotte2, Sylvie Pochet3 and Gilles Labesse4

1 Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France
2 Unité de Biochimie Cellulaire, Institut Pasteur, 75724 Paris Cedex 15, France
3 Unité de Chimie Organique, Institut Pasteur, 75724 Paris Cedex 15, France
4 Centre de Biochimie Structurale, Faculté de Pharmacie, Université de Montpellier I, 34000 Montpellier, France

Reprint requests to: Héléne Munier-Lehmann, Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 28, Rue du Dr Roux, 75724 Paris Cedex 15 - France; e-mail: hmunier{at}pasteur.fr; fax: 33 1 40 61 39 63.

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.

Keywords: Tuberculosis; circular dichroism; fluorescence spectroscopy; molecular modeling; structure-function relationship

Abbreviations: TMPK, thymidylate kinase • TMPKec, thymidylate kinase from E. coli • TMPKmt, thymidylate kinase from M. tuberculosis • TMPKy, thymidylate kinase from yeast • NMPK, nucleoside monophosphate kinase • AZT-MP, 3'-azido-3'-deoxythymidine monophosphate


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