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| Author(s): |
Antonio Marcos Saraiva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2009-09-21 |
| Examining board members: |
Anete Pereira de Souza;
Pietro Ciancaglini;
Marco Aurélio Takita;
Jörg Kobarg;
João Alexandre Ribeiro Gonçalves Barbosa
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| Advisor: | Anete Pereira de Souza |
| Abstract | |
The 9a5c strain from bacterium Xylella fastidiosa was the first phytopathogen to have its genome completely sequenced, which revealed a lot of information about its metabolism and its pathogenicity. 'From a variety of orfs encoded by this bacterium, we highlight, in this work, the XF0703, which correlated protein (with 28.3 kDa) has similarity with SurE proteins from several other bacteria. The SurE proteins *are nucleotidases that dephosphorylate various monophosphorylated nucleosides to their respective nucleosides. This function is critical for maintaining the balanced pool of four (deoxy) ribonucleosides for DNA and RNA synthesis. In this work, we describes the cloning of the XF0703 orf into the vector pET29a, the recombinant protein overexpression (XfSurE) in Escherichia coli BL21(DE3) and the protein purification by nickel affinity chromatography. The secondary structure analysis was done by circular dichroism, while oligomeric state determination was achieved by gel filtration chromatography and small-angle X-ray light scattering (SAXS), which showed that the protein is a tetramer. Functional characterization data indicate that the protein has a highest activity at neutral pH in the presence of manganese as a cofactor, with a highest affinity for the 3-AMP substrate (K0,5 = 0,16 mM). Furthermore, kinetic tests showed that the protein has a allosteric behavior with a high positive cooperativity (Hill coefficient around 2.6) for all natural substrates screened (3-AMP, 5'-dAMP, 5'-AMP and 5'-GMP). Experiments with SAXS technique have allowed to calculate the radius of gyration (32.7 ± 0.2 A), maximum intramolecular distance (100 A) and molecule symmetry. (AU) | |