title: Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation.
abstract: Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 A resolution), the UDP product (2.6 A), or UTP (2.45 A). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.
authors.affiliation: Unité de Chimie Biologique, UMR 206 Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, URA 2171 CNRS, Institut Pasteur, 75724 Paris Cedex 15. briozzo@grignon.inra.fr
authors.forename: Pierre
authors.initials: P
authors.lastname: Briozzo
authors.forename: Cécile
authors.initials: C
authors.lastname: Evrin
authors.forename: Philippe
authors.initials: P
authors.lastname: Meyer
authors.forename: Liliane
authors.initials: L
authors.lastname: Assairi
authors.forename: Nathalie
authors.initials: N
authors.lastname: Joly
authors.forename: Octavian
authors.initials: O
authors.lastname: Barzu
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Gilles
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003461:Crystallography; D004789:Enzyme Activation / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013912:Threonine / Q000235:genetics; D014530:Uridine Diphosphate / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D008024:Ligands; D010710:Phosphates; D013912:Threonine; D014530:Uridine Diphosphate; D001216:Asparagine; D006160:Guanosine Triphosphate; D014542:Uridine Monophosphate; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; D014544:Uridine Triphosphate