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| Author(s): |
Lívia Maria Faim
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2014-04-24 |
| Examining board members: |
Otavio Henrique Thiemann;
José Ramon Beltran Abrego;
Fernanda Canduri;
José Ribamar dos Santos Ferreira Júnior;
Fernando Alves de Melo
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| Advisor: | Otavio Henrique Thiemann |
| Abstract | |
The synthesis and incorporation of selenocysteine in selenoproteins occurs cotranslationally directed by the UGA stop codon. An unique machine of enzymes and protein factors are required for selenocysteine synthesis and decoding of UGA translation termination codon for the insertion of selenocysteine. Among the enzymes involved, Selenonofosfato synthetase (SPS2) is the responsible for catalyzing the activation of selenite with adenosine 5\' - triphosphate (ATP) to generate selenophosphate, the reactive selenium donor, which is substrate of the next pathway enzyme to formation of selenocysteine. Recent studies have identified the presence of selenocysteine biosynthesis in parasites Kinetoplastidas and subsequently, the SPS2 protein of Trypanosoma brucei and Leishmania major have been characterized, however, structural and functional studies of enzymes remain not reported. Thus, this present work report biochemical and biophysical studies of SPS2. To characterize the protein in solution, there were employed the techniques of size exclusion chromatography, native gel electrophoresis, dynamic light scattering (DLS), Small angle X-ray scattering angle (SAXS) and analytical ultracentrifugation (AUC). The results revealed a mixture of dimmers and tetramers in solution for SPS2 with predominance of dimers. Many strategies and improvements in crystallization and diffraction were used to obtain suitable SPS2 crystals for determination of the crystallography structure. T. brucei SPS2 crystals and L. major SPS2 crystals with truncated N-terminal were obtained. However, only the structure of SPS2 protein from L. major with truncated N-terminal to 1.9 Å of resolution was solved. Comparative studies of this structure with other selenophosphate synthases revealed the same structural organization. Functional complementation experiments of truncated and mutated SPS2 revealed three residues located in the SPS2 N- terminal as essential for the activity of the enzyme (Leu33 , Thr34 and Tyr36 to T. brucei SPS2; Leu37 , Thr38 and Tyr40 to L. major SPS2) . Mutational analysis based on the crystal structures indicated that these residues may be involved in the mechanism of selenophosphate delivery to the pathway enzyme next, the selenocysteine synthase. This found could prevent the diffusion of reactive selenium, resulting in selenocysteine synthesis efficient. The results presented here provided important information and new insights about the of selenophosphate synthetase catalysis mechanism in the selenocysteine synthesis pathway. (AU) | |
| FAPESP's process: | 07/06591-0 - Molecular characterization of the selenocisteine synthesis pathway: structural and functional investigation of the human SECp43 |
| Grantee: | Lívia Maria Faim |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |