TAILIEUCHUNG - Báo cáo khoa học: Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase

Bacterial CMP kinases are specific for CMP and dCMP, whereas the rela-ted eukaryotic NMP kinase phosphorylates CMP and UMP with similar efficiency. To explain these differences in structural terms, we investigated the contribution of four key amino acids interacting with the pyrimidine ring of CMP (Ser36, Asp132, Arg110 and Arg188) to the stability, catalysis and substrate specificity ofEscherichia coliCMP kinase. | ỊFEBS Journal Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase Augustin Ofiteru1 Nadia Bucurenci1 Emil Alexov2 Thomas Bertrand3 z Pierre Briozzo3 Helene Munier-Lehmann4 and Anne-Marie Gilles5 1 Laboratory of Enzymology and Applied Microbiology Cantacuzino Institute Bucharest Romania 2 Department of Physics and Astronomy Clemson University SC USA 3 UMR INRA-AgroParisTech 206 de Chimie Biologique Institut NationalAgronomique Paris-Grignon Thiverval-Grignon France 4 Unite de Chimie Organique Institut Pasteur Paris France 5 Unite de Genetique des Genomes Bacteriens Institut Pasteur Paris France Keywords CMP kinase nucleobase specificity protein stability site-directed mutagenesis X-ray crystallography Correspondence . Gilles Unite de Genetique des Genomes Bacteriens Institut Pasteur 28 rue du docteur Roux 75724 Paris France Fax 33 1 45 68 89 48 Tel 33 1 45 68 89 68 E-mail amgilles@ Present address Sanofi-Aventis ChemicalSciences Vitry-sur-Seine France Received 5 February 2007 revised 2 May 2007 accepted 7 May 2007 doi Bacterial CMP kinases are specific for CMP and dCMP whereas the related eukaryotic NMP kinase phosphorylates CMP and UMP with similar efficiency. To explain these differences in structural terms we investigated the contribution of four key amino acids interacting with the pyrimidine ring of CMP Ser36 Asp132 Arg110 and Arg188 to the stability catalysis and substrate specificity of Escherichia coli CMP kinase. In contrast to eukaryotic UMk CMP kinases which interact with the nucleobase via one or two water molecules bacterial CMP kinase has a narrower NMP-binding pocket and a hydrogen-bonding network involving the pyrimidine moiety specific for the cytosine nucleobase. The side chains of Arg110 and Ser36 cannot establish hydrogen bonds with UMP and their substitution by hydrophobic amino acids simultaneously affects .

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