|PDF Version||Macromolecule Structural Chemistry|
|Director : BÂRZU Octavian (firstname.lastname@example.org)|
Our research activity is focused on kinases from pathogenic organisms using nucleotides or sugars as substrates and which represent potential targets for antibacterial therapy. Two enzymes from Mycobacterium tuberculosis and Streptococcus pneumoniae were explored with particular interest this year. Several genes encoding proteins of unknown function from M. tuberculosis were expressed for structural analysis.
1. Nucleoside monophosphate (NMP) kinases from M. tuberculosis(H. Munier-Lehmann in collaboration with G. Labesse, D. Douguet, L. Dugué, O. Dutruel, S. Pochet, P. Herdewijn, S. Van Callenbergh, A. Haouz, P. Alzari)
Our aim is the synthesis of new specific inhibitors directed against mycobacteria by structure-based (X-ray or NMR) design. Two NMP kinases have been selected, TMP kinase and UMP kinase. Both enzymes exhibit significant differences when compared to their eukaryote counterparts. This work was financed by a Transversal Research Program, joining different laboratories on the Campus. We pursued our previous program concerning thymidine analogs as competitive inhibitors of M. tuberculosis TMP kinase. Furthermore we selected by in silico screening a number of non-nucleoside compounds acting of TMP kinase at mM concentrations. New bi-substrate analogs will be also synthesized and explored, the best inhibitors being submitted to co-crystallization studies with the bacterial enzyme. The biochemical characterization of UMP kinase from M. tuberculosis indicated specific mechanisms of activation by GTP and inhibition by UTP. Crystals diffracting at 3.5 Å were obtained. They allowed determination of the quaternary structure, but the architecture of the active site was not yet determined.
2. UMP kinases from Hemophilus influenzae and Streptococcus pneumoniae(A.-M. Gilles, L. Assairi, C. Evrin, P. Christova, M. Ionescu, I.O. Sirbu, N. Slavova-Azmanova, in collaboration with N. Bucurenci, G. Labesse and C.T. Craescu)
Bacterial NMP kinases are unrelated to eukaryote UMP/CMP kinases both at the level of primary and three-dimensional structure. A model of UMP kinase from E. coli and B. subtilis was proposed, based on the carbamate kinase fold conservation. The active and the allosteric site of UMP kinase from two pathogenic organisms was explored by using fluorescent markers (3'-Ant-dGTP, 3'-Ant-dUTP and 3'-Ant-dADP). These studies indicated that the activating and the inhibiting sites are largely overlapping. Isothermal calorimetry substantiated the spectroscopic analysis.
3. deoK operon (A.-M. Gilles, L. Assairi in collaboration with C.T. Craescu, T. Bertrand, P. Briozzo, C. Le bouguenec and J. Neuhard)
Salmonella enterica, contrary to E. coli K12 is capable to grow on deoxyribose (dR) as sole source of carbon and energy, a property which is conferred by a specific deoxyribokinase (DeoK). The deoK gene as well as two other genes, deoP and deoX form an operon (deoKPX) which is controlled by a specific repressor (DeoQ). We showed that deoQ and deoKPX operon are transcribed by two different promoters located between deoQ and deoK genes. The promoter of deoKPX operon is ten fold stronger than the deoQ promoter. We also demonstrated that deoK operon is present in Citrobacter freundii, Yersinia enterolitica, Agrobacterium tumefaciens and several pathogenic strains of E. coli. In collaboration with C. Le bouguenec we explored this last aspect which was financed by the Transversal Research Program 53. We also explored the putative function of DeoX which is required for an optimal bacterial growth on dR. Pairwise sequence alignment of DeoX with GalM and XylM, whose biochemical function is the interconversion of the a and b anomers of several carbohydrates indicated identity varying from 12.5 to 15.1%. We tested DeoM for mutarotase activity on various sugars and found its high specificity for dR as substrate. By site-directed mutagenesis we identified some key residues essential for catalysis. DeoX was called DeoM by analogy to GalM.
4. NAD kinase from B. subtilis(L. Assairi in collaboration with G. Labesse and D. Douguet)
Site-directed mutagenesis of several residues belonging to NAD kinase and conserved in 6-phosphofructokinase from E. coli indicated that the GGDGT motif is involved in ATP recognition. Several modified formed of B. subtilis NAD kinase (G44A, D45A and G46) were found indeed catalytically inactive. The T47A mutant although still active with ATP is devoid of phosphate transfer capacity when UTP or CTP are used as donors. Another residue recognized as important for catalysis is Phe74. NAD kinase is an interesting candidate for structural analysis by X-ray.
5. Structural genomics at the Pasteur Institute(L. Assairi, H. Munier-Lehmann and E. Seclaman)
Our laboratory participates to the project of M. tuberculosis structural genomic. We cloned and overexpressed 15 genes encoding proteins of unknown function. The majority of these proteins expressed in E. coli were found insoluble, thus requiring particular culture conditions to increase the yield of soluble forms. One of these proteins was found stable and adapted for crystallization and structural analysis.
Keywords: kinases, pathogenic organisms, deo operon, conception of inhibitors, structural genomic
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|LAMBRECHT, Marie-Régine, Secretary,email@example.com||BÂRZU Octavian, CNRS, Research Director, 1st class, Head of the firstname.lastname@example.org
GILLES Anne-Marie, INSTITUT PASTEUR, Laboratory Chief.email@example.com
MUNIER-LEHMANN Hélène, INSERM, Research Assistant 1st firstname.lastname@example.org
|ASSAIRI Liliane, CNRS, Research assistant.
CHRISTOVA Petya, Researcher.
EVIN Cécile, DEA Student.
LEON Ruth, PhD student.
SECLAMAN Edward, Researcher.
SIRBU Ioan, Ovidiu, Researcher.
SLAVOVA-ASMANOVA Neli, Researcher
|Lambrecht Marie-Régine, Secrétaire,email@example.com|