Bacterial arsenite oxidation at the molecular level

• Autor(a) principal: Engrola, Filipa Sofia Silva
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/89008
• topic: Arsenic; antimony; arsenite oxidase; molybdoenzymes; X-ray crystallography; microscale thermophoresis; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

According to the WHO, arsenic is one of the top 10 chemical contaminants in drinking-water worldwide and affects more than 140 million people. The arsenite oxidising enzyme (Aio), from microorganisms Rhizobium sp. NT-26 (NT-26_Aio) and Alcaligenes faecalis (A.f._Aio), and their final electron acceptors – cytochrome c552 (NT-26_cytc552) and azurin (A.f._azu), respectively – are currently being studied for their use as biosensors and in bioremediation processes. Both Aio enzymes share high structural similarity (948 matching residues with an r.m.s.d. of 1.84 Å for Cα atoms) and are composed of a large subunit (AioA) which contains a molybdenum centre and a [3Fe-4S] cluster, and a small subunit (AioB) that possess a Rieske [2Fe-2S] cluster. Aiming to elucidate the catalysis mechanism of the enzymes, and their electron transfer to the final electron acceptors, a combination of expression and purification of the proteins, crystallisation, structural analysis, enzyme kinetics and affinity tests were conducted. A 1.84 Å resolution structure of A.f._Aio in complex with a substrate analogue - SbV oxoanion - was determined using molecular replacement (PDB: IG8K). Additionally, a previously obtained 1.89 Å resolution structure of NT-26_Aio, containing a SbIII oxoanion near the active site, was investigated and used for comparison. Analysis of bond lengths and geometry of the ligands at the Mo active site suggests that both crystallized enzymes reveal different reaction intermediates, corresponding to different stages of the mechanism. The specific activity of two active site mutants of NT-26_Aio – D169A and E453A – determined by UV-Vis spectroscopy, revealed that these only uphold 46 and 8% of the WT enzyme efficiency, respectively. This information, together with structural analysis, strongly suggest that both amino acid residues play an essential role in substrate orientation through a complex network of hydrogen-bonds.