tailieunhanh - Báo cáo khoa học: Structural and functional investigations of Ureaplasma parvum UMP kinase – a potential antibacterial drug target

The crystal structure of uridine monophosphate kinase (UMP kinase, UMPK) from the opportunistic pathogenUreaplasma parvumwas deter-mined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family. | ỊFEBS Journal Structural and functional investigations of Ureaplasma parvum UMP kinase - a potential antibacterial drug target Louise Egeblad-Welin1 Martin Welin2 z Liya Wang1 and Staffan Eriksson1 1 Department of Anatomy Physiology and Biochemistry Swedish University of AgriculturalSciences Uppsala BiomedicalCentre Sweden 2 Department of Molecular Biology Swedish University of AgriculturalSciences Uppsala BiomedicalCentre Sweden Keywords bacterialUMP kinase Mollicutes mycoplasma subunit interaction Ureaplasma parvum Correspondence S. Eriksson Department of Anatomy Physiology and Biochemistry Swedish University of AgriculturalSciences Box 575 BiomedicalCenter S-751 23 Uppsala Sweden Fax 46 18550762 Tel 46 184714187 Email Present address StructuralGenomics Consortium Karolinska Institutet Stockholm Sweden Database The structure has been submitted to the Protein Data Bank under the accession number 2va1 The crystal structure of uridine monophosphate kinase UMP kinase UMPK from the opportunistic pathogen Ureaplasma parvum was determined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family. Recombinant UpUMPK exhibited Michaelis-Menten kinetics with UMP with Km and Vmax values of 214 4 pM and 262 24 pmol-min-1-mg_1 respectively but with ATP as variable substrate the kinetic analysis showed positive cooperativity with an n value of . The end-product UTP was a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP. Unlike UMPKs from other bacteria which are activated by GTP GTP had no detectable effect on UpUMPK activity. An attempt to create a GTP-activated enzyme was made using site-directed mutagenesis. The mutant enzyme F133N F133 corresponds to the residue in Escherichia coli that is involved in GTP activation with F133A as a control were expressed purified and characterized. Both enzymes exhibited negative cooperativity with UMP and .