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Structural studies on molybdenum-dependent enzymes: from transporters to enzymes
Publication . Cardoso, Ana Rita Castro Otrelo; Silva, Teresa; Romão, Maria João
Molybdenum (Mo) and tungsten (W) are heavy metals that can be found in the active site of several enzymes important for the metabolism of carbon, sulfur and nitrogen compounds. This Thesis describes the structural studies of two proteins that are involved in Mo and W uptake (TupA and ModA), of a Mo-containing aldehyde oxidoreductase (PaoABC) and of its chaperone PaoD. The main techniques used for the structural characterization of these proteins are X-ray crystallography and Small-Angle X-ray Scattering (SAXS), which are presented in Chapter 1, including a brief introduction about the importance of Mo and W in biological systems. Mo or W cofactor biosynthesis requires the presence of molybdate and tungstate inside the cells, which is achieved by specific ABC transport systems. Chapter 2 presents a small introduction about these transport systems, followed by the structural characterization and analysis of ModA and TupA from Desulfovibrio alaskensis G20. The tridimensional structures were determined by X-ray crystallography and SAXS, and the implication in the molybdate/tungstate uptake and discrimination between ligands discussed. The results show that TupA has a high selectivity for tungstate, while ModA is not able to distinguish between the two oxyanions. An important residue for TupA selectivity was identified, R118, paving the way for future biotechnological applications. Chapter 3 focuses on Mo-containing enzymes and cofactor maturation. The tridimensional structure of the Escherichia coli periplasmic aldehyde oxidoreductase PaoABC was solved at 1.7 Å resolution, revealing the presence of an unexpected [4Fe-4S] cluster that was not previously reported. The PaoABC structure has unique features, being the first example of an heterotrimer (αβγ) from the xanthine oxidase family. The activation of PaoABC is dependent on its interaction with the chaperone PaoD, which was also studied. The stabilization of E. coli PaoD is extremely challenging but the results here presented show that the presence of ionic liquids during thawing avoids protein aggregation. This allowed the identification of two promising crystallization conditions using polyethylene glycol and ammonium sulfate as precipitant agents. Chapter 4 describes the use of SAXS for the characterization of a multi-component biosensor to detect chronic myeloid leukemia, demonstrating the versatility of this technique to determine the envelope of biological molecules as oligonucleotides. The main conclusions derived from the work here described, as well as future perspectives, are drawn in Chapter 5.
The first mammalian aldehyde oxidase crystal structure: Insights into substrate specificity
Publication . Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João; DQ - Departamento de Química; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); ASBMB - American Society for Biochemistry and Molecular Biology
Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9A? . This is the first mammalian AOX whose structure has been solved. The structureprovidesimportantinsightsintotheproteinactivecenter and further evidence on the catalytic differences characterizing AOXand xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.
Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2.
Publication . Marangon, Jacopo; Correia, Hugo D.; Brondino, Carlos D.; Moura, José João Galhardas de; Romão, Maria João; Gonzalez, Pablo Javier; Santos-silva, Teresa Sacadura; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); DQ - Departamento de Química; PLOS - Public Library of Science
Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-PRO/118377/2010