Avaliação bioquímica e estrutural de enzimas recombinantes envolvidas na degradação de hidrocarbonetos aromáticos policíclicos

Abstract

Polycyclic Aromatic Hydrocarbons (PAHs), organic compounds formed by fused benzene rings, are environmental contaminants that occur naturally during the combustion of organic matter as well as by oils spills and other industrial processes. There is a great diversity of bacteria capable of degrading the PAHs and the enzymes present in the metabolic pathways can be used for the bioremediation of these pollutants. Naphthalene is one of the most toxic HAPs being used as a model in the degradation study of these compounds and in P. putida G7 the metabolism of naphthalene is a well-studied process. The enzymatic complex of naphthalene dioxygenase (NahA) acts in the first reaction of the degradation pathway of this contaminant and NahA is a member of a large family of Rieske oxygenases which initiates the degradation of various aromatic compounds. However, the search for potential enzymes involved in the degradation of PAHs is not limited to P. putida G7, and different validated sources can be included to enrich the databases. In this work the search for proteins homologous to NahA was carried out in order to obtain a dioxygenase complex that could act on several aromatic substrates. The gene sequences of the dioxygenase complex proteins Aa, Ab, Ac and Ad from Achromobacter xylosoxidans A8 were selected from genomic and metagenomics databases of different organisms. Recombinants proteins were expressed separately in three components (Aa, Ab and AcAd) as well as co-expressed together and after the expression, cells were lysed for protein release and the resulting extract was immediately subjected to purification by affinity chromatography followed by a size exclusion chromatography. The purification process with the system containing the dioxygenase complex (AaAbAcAd) was the most satisfactory and therefore it was decided to proceed with this sample. Recombinant proteins were identified by western blotting and mass spectrometry. Dynamic light scattering (DLS) assays with the samples showed the proteins presented in solution as monodisperse, with Aa as monomeric and AcAd as hexameric forms.The enzymatic activity of the complex was measured by the NADH consumption. Crystallographic structure of the AcAd proteins was solved at 1.35 Å and the crystallographic model presented an α/β folding typical of the Rieske oxygenases, with the well-defined Rieske [2Fe-2S] and catalytic domains in the alpha subunit, except for the presence of the mononuclear manganese in the active site, rather than iron, a conserved metal of the dioxygenases. In the asymmetric unit, the molecule was found in the form of αβ heterodimer and with crystallographic symmetry operations the biological unit was observed, in the form of heterohexamer (α3β3). Cys-104, His-106, Cys-124 and His-127 residues in the Rieske domain and His-232, His-237 and Asp-379 in the catalytic domain are conserved residues for all Rieske oxygenases being important for catalysis. The non-conserved residues present in the catalytic site Leu-325, His-323, Val-375 and Ile-369 proved to be important and could contribute to the specificity and accommodation of substrates. In the crystallographic model containing catechol, it was bound to the active site, interacting with Asn-226. The results of this work present, for the first time, studies of structural characteristics concerning the catalytic component of the dioxygenase complex from Achromobacter xylosoxidans A8, which can contribute for future studies on the identification of key residues for catalysis on several substrates.