Abstracts Category : Other

Structural studies on molybdenum-dependent enzymes: from transporters to enzymes

by Ana Rita Cardoso

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 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 () 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.Advisors/Committee Members: Silva, Teresa, Romo, Maria Joo.