Full text
| |
| Author(s): |
Nathalia de Campos Rodrigues
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2014-01-17 |
| Examining board members: |
Glaucius Oliva;
Marcio Vinicius Bertacine Dias;
Marcos Roberto de Mattos Fontes;
Humberto D\'Muniz Pereira;
Ariel Mariano Silber
|
| Advisor: | Glaucius Oliva |
| Abstract | |
Neglected tropical diseases share features that allow them to persist in conditions of poverty, which clump together and frequently overlap. More than 1 billion people suffer from one or more neglected tropical diseases. Among them is the Chagas disease is an important parasitic disease resulting from infection by the flagellate protozoan parasite Trypanosoma cruzi, with triatomine insects as vectors. Topoisomerases are ubiquitous enzymes involved in the regulation of DNA supercoiling and overcoming topological barriers during replication, transcription, recombination and repair. The type II topoisomerases are essential for trypanosomatids since, in addition to their role in nuclear DNA metabolism, these enzymes might also play an important role in the replication and organization of the DNA contained in the specialized region of the mitochondrion known as the kinetoplast. The study of Mitochondrial Topoisomerase II from T. cruzi (TcTopoIImit) was performed by sequence and structural analyses into topos members for determination of domains constructions for cloning. Constructions for the N-domain were then cloned in pTZ57R/T vector and subcloned into pET system vectors for protein expression in E. coli. The protein expression experiments were performed by different strain cells, vectors, buffers solutions and others adjustable parameters for improve the solubility but recombinant products in soluble form were not obtained. The flavoprotein Old Yellow Enzyme from Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced, purified, crystallized by the vapour diffusion method and its crystallography structure was solved for molecular replacement and refined. TcOYE was crystallized in two crystalline forms, P212121 and P21 and diffraction data were collected to a maximum resolution of 1.27 and 2.00 Å, respectively. The atomic coordinates and structure factors of the TcOYE structure in P212121 and P21 crystalline forms have been deposited in the Protein Data Bank with the accession codes 4E2B and 4E2D, respectively. TcOYE displays a canonical (α/β)8-barrel fold with a FMN prosthetic group located at the large active-site cavity. Afterwards, sequential and structural analyses were carried out for TcOYE and others OYEs. The region 141-156 of the capping subdomain in TcOYE as well as in others OYEs is intrinsically flexible exhibiting high B-factor values. The most divergent region among these OYEs is the extended loop (289-297), which can vary in length and composition changing the volume and accessibility to the active site. (AU) | |