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Please use this identifier to cite or link to this item: http://hdl.handle.net/10362/6700

Título: Crystallographic studies on molybdopterin-dependent enzymes
Autor: Coelho, Catarina Maria Campos
Orientador: Romão, Maria João
Trincão, José
Issue Date: 2011
Editora: Faculdade de Ciências e Tecnologia
Resumo: This Thesis reports the determination of the crystal structure of two molybdenum-dependent enzymes, as well as its functional interpretation. In Chapter 1 is given a general introduction on the use of molybdenum in biological systems, particularly its incorporation into the active site of several enzymes. In the same chapter is also included an overview on X-ray protein crystallography, briefly describing its main basic principles. Aldehyde oxidases are homodimeric proteins belonging to the xanthine oxidase (XO) family of molybdenum containing enzymes. The three-dimensional structure of mouse aldehyde oxidase homologue1 (mAOH1) is here reported and described (Chapter 2). This constitutes the first crystal structure ever obtained for an aldehyde oxidase. The mAOH1 protein was extracted from rat liver, and heterologously expressed in E.coli. The recombinant protein allowed determining suitable crystallization conditions, which were reproduced using the native enzyme from mouse liver. Suitable crystals were obtained, allowing to solve the protein structure at 2.9Å resolution, using bovine milk xanthine oxidase as a search model. Both proteins belong to the XO family of Mo proteins and are very similar, although catalyzing different reactions. The structure of mAOH1 and its comparison with the XO structure allowed drawing important structure and function correlations, and to explain the different enzyme specificities. These studies have also contributed to better understand the role of aldehyde oxidase in human health. The enzyme has received considerable attention from several pharmaceutical companies, as it is involved in the detoxification of several drugs and xenobiotics, assuming particular relevance in human health and drug design studies. Periplasmic nitrate reductase from the Cupriavidus necator bacterium (Cn NapAB) is a heterodimeric protein, and belongs to the DMSO reductase family of molybdenum containing enzymes. The three-dimensional structure of the C.necator NapAB was solved at 1.5Å resolution using crystals obtained from a crystallization robot. Structural, spectroscopic and functional studies of this protein are reported in Chapter 3. The high resolution of the model, allowed identifying the true nature of all Mo ligands. In the first reported nitrate reductase crystal structure (NapA from Desulfovibrio desulfuricans), the 6th Mo ligand had been identified as an oxygen, but in Cn NapAB, a sulfur atom could be unambiguously assigned to this position. It is believed that this is a general feature of all nitrate reductases, which has led to the necessary revisions on the reaction mechanism for this family of enzymes. To further characterize C.necator NapAB, spectroscopic and electrochemical studies have also been performed, and have shown unexpected features, particularly regarding the potential of the two c-type hemes.
Descrição: Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural
URI: http://hdl.handle.net/10362/6700
Appears in Collections:FCT: DQ - Teses de Doutoramento

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