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Anti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidy-late (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed inEscherichia coliafter cloning the gene (A48R). | iFEBS Journal Substrate specificity of vaccinia virus thymidylate kinase Dimitri Topalis1 Bruno Collinet1 Cecile Gasse2 Laurence Dugue2 Jan Balzarini3 Sylvie Pochet2 and Dominique Deville-Bonne1 1 Laboratoire d Enzymologie Moleculaire et Fonctionnelle FRE 2852 CNRS Paris France 2 Unite de Chimie Organique URA 2128CNRS Institut Pasteur Paris France 3 Rega Institute for MedicalResearch Leuven Belgium Keywords antiviralnucleoside thymidylate kinase 5-Iododeoxyuridine MABA derivative poxvirus Correspondence D. Deville-Bonne Laboratoire d Enzymologie Moleculaire et Fonctionnelle FRE 2852 CNRS-Paris 6 T43-44 4e 4 place Jussieu 75251 Paris Cedex 05 France Fax 33 1 44 27 59 94 Tel 33 1 44 27 59 93 E-mail ddeville@ccr.jussieu.fr Received 11 August 2005 revised 29 September 2005 accepted 5 October 2005 doi 10.1111 j.1742-4658.2005.05006.x Anti-poxvirus therapies are currently limited to cidofovir S -1- 3-hydroxy-2-phosphonylmethoxypropyl cytosine but drug-resistant strains have already been characterized. In the aim of finding a new target the thymidylate TMP kinase from vaccinia virus the prototype of Orthopoxvirus has been overexpressed in Escherichia coli after cloning the gene A48R . Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42 identity with human TMP kinase the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2 and dTMP were 0.15 mM and 20 pM respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2 -deoxyuridine 5 -monophosphate 5I-dUMP and 5-bromo-2 .