title: UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.
abstract: The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.
authors.affiliation: Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, Paris, France.
authors.forename: Cristina
authors.initials: C
authors.lastname: Gagyi
authors.forename: Nadia
authors.initials: N
authors.lastname: Bucurenci
authors.forename: Ovidiu
authors.initials: O
authors.lastname: Sîrbu
authors.forename: Gilles
authors.initials: G
authors.lastname: Labesse
authors.forename: Mihaela
authors.initials: M
authors.lastname: Ionescu
authors.forename: Augustin
authors.initials: A
authors.lastname: Ofiteru
authors.forename: Liliane
authors.initials: L
authors.lastname: Assairi
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Landais
authors.forename: Antoine
authors.initials: A
authors.lastname: Danchin
authors.forename: Octavian
authors.initials: O
authors.lastname: Bârzu
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Gilles
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; C078379:pyrH protein, E coli; D006160:Guanosine Triphosphate; D014166:Transferases; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase