title: Substrate specificity of vaccinia virus thymidylate kinase.
abstract: 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 microM, 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'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.
authors.affiliation: Laboratoire d'Enzymologie Moléculaire et Fonctionnelle, FRE 2852 CNRS, Paris, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Topalis
authors.forename: Bruno
authors.initials: B
authors.lastname: Collinet
authors.forename: Cécile
authors.initials: C
authors.lastname: Gasse
authors.forename: Laurence
authors.initials: L
authors.lastname: Dugué
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
authors.forename: Sylvie
authors.initials: S
authors.lastname: Pochet
authors.forename: Dominique
authors.initials: D
authors.lastname: Deville-Bonne
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular / Q000379:methods; D003854:Deoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D016415:Sequence Alignment; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase