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J. Biol. Chem., Vol. 280, Issue 27, 25533-25540, July 8, 2005

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Structure of Escherichia coli UMP Kinase Differs from That of Other Nucleoside Monophosphate Kinases and Sheds New Light on Enzyme Regulation^*

Pierre Briozzo, Cécile Evrin¶, Philippe Meyer||**, Liliane Assairi, Nathalie Joly, Octavian Brzu¶, and Anne-Marie Gilles¶

From the Unité de Chimie Biologique, UMR 206 Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, the^¶ Unité de Génétique des Génomes Bactériens, URA 2171 CNRS, Institut Pasteur, 75724 Paris Cedex 15, the ^||Laboratoire d'Enzymologie et de Biochimie Structurales, UPR 9063 du CNRS, 91198 Gif-sur-Yvette Cedex, and the Institut Curie, Centre Universitaire Paris-Sud, 91405 Orsay, France

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 Å resolution), the UDP product (2.6 Å), or UTP (2.45 Å). 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.

Received for publication, February 18, 2005 , and in revised form, March 30, 2005.

^* This work was supported by grants from the Centre National de la Recherche Scientifique (Grants UPR 9063, URA 2185, and URA 2171), the Institut National de la Recherche Agronomique (Grant UMR 206), the Institut Pasteur (Grant AC02), and AstraZeneca R & D, Boston, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2BNE, 2BND, 2BNF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

To whom correspondence may be addressed. Tel.: 33-1-30-81-54-73; Fax: 33-1-30-81-53-73; E-mail: briozzo{at}grignon.inra.fr. ** A postdoctoral fellow of the Association pour la Recherche contre le Cancer. To whom correspondence may be addressed. Tel.: 33-1-69-82-42-49; Fax: 33-1-69-82-31-29; E-mail: meyer{at}lebs.cnrs-gif.fr.

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