Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase

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Abstract

Bacterial UMP kinases do not exhibit any sequence homology with other nucleoside monophosphate kinases described so far, and appear under oligomeric forms, submitted to complex regulation by nucleotides. We propose here a structural model of UMP kinase from Escherichia coli based on the conservation of the fold of carbamate kinase whose crystal structure was recently solved. Despite sequence identity of only 18% over 203 amino acids, alignment of UMP kinase from E. coli with carbamate kinase from Enterococcus faecalis by hydrophobic cluster analysis and threading suggested the conservation of the overall structure, except for a small subdomain (absent in UMP kinase). The modelled dimer suggested conservation of the dimer interface observed in carbamate kinase while interaction of UMP kinase with a monoclonal antibody (Mab 44-2) suggests a three in-plane dimer subunit arrangement. The model was analyzed in light of various modified forms of UMP kinase obtained by site-directed mutagenesis.

Section snippets

Materials and methods

Chemicals. Nucleotides, restriction enzymes, T4DNA ligase, and coupling enzymes were from Roche-Diagnostics, Mannheim, Germany. T7DNA polymerase and the four deoxynucleoside triphosphates used in sequencing reactions were from Pharmacia. Oligonucleotides were synthesized by the phosphoamidinate method. NDP kinase from Dictyostelium discoideum (2000 U/mg prot) was provided by M. Véron. Mab 44-2 was obtained by immunizing mice with a C-terminal truncated form of E. coli UMP kinase (amino acids

Sequence analysis of bacterial UMP kinases

Database screening using the program PSI-BLAST with UMP kinase from E. coli as a query first showed sequence similarities with UMP kinases from other bacteria (probability scores: e-66 to e-53). Various other bacterial kinases followed immediately after, in the PSI-BLAST output (pyrroline-5 carboxylate synthase with probability scores: e-49 to e-44; glutamate-5-kinase, e-43 to e-41; aspartokinases, e-41 to e-31 and carbamate kinases, e-30 to e-23). No similarity with eukaryotic UMP kinases was

Acknowledgements

This work was supported by grants from the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale (U554, the Centre National de la Recherche Scientifique (URA 2185 and UMR 5048), the Genopole Languedoc-Roussillon, and AstraZeneca R&D Boston Inc. We thank Pedro Alzari and Vicente Rubio for many fruitful discussion, Susan Michelson for critical comments, and Régine Lambrecht for excellent secretarial help.

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    Abbreviations: CKef, Carbamate Kinase from Enterococcus faecalis; CPSpf, Carbamoyl Phosphate Synthase from Pyrococcus furiosus. The one-letter code is used for amino acids.

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