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  Director : BÂRZU Octavian (obarzu@pasteur.fr)


  abstract

 

Our research activity is focused on kinases acting on nucleotides or sugars as substrates and which are potential targets for antibacterial therapy. In the structural genomic project of the Pasteur Institute to which our laboratory is associated, we have cloned a number of ten genes from M. tuberculosis and coding for proteins with unknown, or putative function. Part of them were overproduced in E. coli for structural analysis.



  report

cale

1. Nucleoside monophosphate (NMP) kinases from M. tuberculosis

(H. Munier-Lehamnn in collaboration with G. Labesse, D. Douguet, L. Dugué, S. Pochet, P. Herdewijn, S. Van Callenbergh, B. Gomes-Guimaraes and P. Alzari)

One of the aims of our research projects is the obtention of new specific inhibitors directed against mycobacteria by structure-based (X-ray crystallography or nuclear magnetic resonance spectroscopy) design. Two NMP kinases have been selected for their particular properties as compared to their eukaryote homologs, TMP- and UMP kinase. This work is the subject of one Transversal Research Programm joining different laboratories of the Pasteur Institute. In this year, about 80 analogs of natural substrate (dTMP) have been tested in vitro as inhibitors of TMP kinase from M. tuberculosis. Half on these compounds have been synthesized by the group of S. Pochet from the Unit of Organic Chemistry. Five of these molecules exhibiting an inhibition constant (Ki) in the micromolar range will be tested on cultures from M. tuberculosis.

With concerns UMP kinase, the three-dimensional structure of the bacterial enzyme was not yet resolved. UMP kinase from M. tuberculosis is a good candidate for X-ray studies due to its better stability and solubility in comparison with other UMP kinases isolated and characterized in our laboratory. Crystals of UMP kinase from M. tuberculosis obtained last year with UTP, have diffracted at low resolution (7-8 Å). A new construct of UMP kinase from M. tuberculosis with a shorter tag sequence has been obtained. A better quality of crystals was obtained with this new protein both in the presence and absence of GTP. We hope to get soon resolution at < 2 Å to be latter used for search of new inhibitors of UMP kinase from M. tuberculosis.

2. NMP kinases from other bacterial species

(A.-M. Gilles, A. Ofiteru, C. Gagyi, H. Munier-Lehmann in collaboration with T. Bertrand, P. Briozzo)

The substitution, by site-directed mutagenesis, of residues involved in the interaction of CMP kinase from E. coli with the cytosine moiety of CMP (Ser36, Asp132, Arg110 and Arg188) was shown to affect considerably the Km and the rate of NMP phosphorylation. On the contrary, the kinetic parameters concerning the donor substrate, ATP, are similar to the wild-type enzyme. The Arg188Met variant whose stability is the same as that of the wild-type enzyme has been crystallized alone or in complex with the natural substrates (CMP or dCMP). We showed that the pocket accomodating the NMP was enlarged by Arg188 ® Met substitution preventing in this way the interaction of the enzyme with the sugar and the phosphate group of its substrate. The structural approach allowed us a better evaluation of the consequences generated by aminoacid substitution on the network of hydrogen bonds essential for catalysis.

We have pursued our work on the biochemical characterization of several bacterial UMP kinases part of them from pathogenic organisms. These experiments demonstrated the heterogeneity of different preparations due to the presence of aggregates, a drawback for the obtention of reliable crystals. One variant of UMP kinase from E. coli (Asp159Asn), more soluble and homogenous, is a good candidate for X-ray crystallography.

3. Other targets for antibacterial agents

(L. Assairi in collaboration with G. Labesse and D. Douguet)

NAD kinase phosphorylates NAD to NADP which is a cofactor for many biosynthetic cellular reactions. Several differences have been observed between eukaryotes and prokaryotes concerning the specificity of the phosphate donor. Molecular modeling studies have shown the relationship between NAD kinases and 6-phosphofructokinase from E. coli. The presence of a conserved sequence in both enzymes (GGDGT) has prompted us to replace by site-directed mutagenesis the Asp ® Ala residue in NAD kinase from N. meningitidis. The variant in which Asp is substituted with an Ala residue is inactive.

We have also selected the dephosphoCoa kinase as a new target. The bacterial enzyme catalyzes the final steps in the biosynthesis of coenzyme A. We have cloned the coaE gene coding for dephosphoCoa kinase from N. meningetidis (NMA2157) and from M. tuberculosis and presently we characterize the recombinant proteins expressed in E. coli.

4. deoK operon from Salmonella typhimurium

(A.-M. Gilles, J. Ferdinand, C. Gagyi, L. Assairi in collaboration with C. Le Bouguenec, T. Bertrand, P. Briozzo and J. Neuhard)

The Salmonella species contrary to E. coli K12 are able to use deoxyribose (dR) as a sole carbon and energy source . We have demonstrated that this property was due to the presence of three genes encoding for a permease (deoP), a desoxyribokinase (deoK) and a protein of yet unknown function (deoX), respectively. These genes form an operon (deoK) whose expression is regulated by a repressor (DeoQ). Analyses by two-dimensional gel electrophoresis of extracts from S. typhimurium, grown in the presence or in the absence of dR, have demonstrated the induction of the synthesis of these proteins, together with the four enzymes of an other operon called deo. Searches for the deoK operon in gene and protein databanks have shown that the deoK operon is incomplete in Agrobacterium tumefaciens and Rhodobacter spheroides species. The deoK operon is active in Citrobacter freundii species and in some pathogenic strains of E. coli, associated with pathogenicity islands. In last case, we have shown that the deoK operon conserves all its properties, as found initially in Salmonella, i.e. the capacity to utilize dR for multiplication. Biochemical studies of DeoX showed that this stable hexamer protein forms crystals diffracting at 2.7. The selenomethionylated protein has recently been produced to facilite the resolution of its three-dimensional structure.

5. Structural genomics at the Pasteur Institute

(L. Assairi, H. Munier-Lehmann and D. Portnoï)

In the structural genomics project in which our laboratory is involved, we started the cloning of ten different genes from M. tuberculosis, encoding for proteins of unknown functions and having no similarity with proteins of origin different than that of mycobacteria or actinomycetes. In collaboration with the Unit of Structural Biochemistry, these proteins will be used for crystallogenesis assays in view of determining their three-dimensional structure. Biochemical studies will be undertaken in parallel to determine their function, with the hope that some will be used also as potential targets in antituberculosis therapy.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

LAMBRECHT, Marie-Régine, lambrech@pasteur.fr

BÂRZU, Octavian, CNRS, Research Director 1st class, Head of the laboratory.

GILLES, Anne-Marie, IP, Laboratory chief.

MUNIER-LEHMANN, Hélène, INSERM, Research assistant.

PORTNOI, Denis, IP, Research assistant.

ASSAIRI, Liliane, CNRS.

GAGYI, Elena, University of Medicine and Pharmacy, Cluj-Napoca, Romania.

IONESCU, MihaelaUniversity of Medicine and Pharmacy, Cluj-Napoca, Romania.

OFITERU, Augustin, Cantacuzene Institute, Bucharest, Romania.

SIRBU, Ioan, University of Medicine and Pharmacy, Timisoara, Romania.

FERDINAND, Joelle, DEA student.

LAMBRECHT, Marie-Régine, Secretary, lambrech@pasteur.fr


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