dc.contributorRonaldo Alves Pinto Nagem
dc.contributorCarlos Basilio Pinheiro
dc.contributorTiago Antonio da Silva Brandao
dc.contributorAlexsandro sobreira Galdino
dc.contributorIeso de Miranda Castro
dc.creatorDebora Maria Abrantes Costa
dc.date.accessioned2019-08-14T09:59:15Z
dc.date.accessioned2022-10-04T01:04:03Z
dc.date.available2019-08-14T09:59:15Z
dc.date.available2022-10-04T01:04:03Z
dc.date.created2019-08-14T09:59:15Z
dc.date.issued2014-05-13
dc.identifierhttp://hdl.handle.net/1843/BUBD-A9NLVA
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/3838550
dc.description.abstractThe three dimensional structure of enzymes allows us to determine their action mechanism, to act in a targeted manner in the structure of these molecules and eventually change physicochemical properties in favor of any specific biotechnological applications, and contribute to a basic understanding of the proteins structure-function. cis-Naphthalenedihidrodiol dehydrogenase (NahB) and salicylate hydroxylase (NahG) enzymes from Pseudomonas putida G7 are involved in degradation of naphthalene, a polycyclic aromatic hydrocarbon (PAH). Most PAHs are toxics, mutagenics and carcinogenics. Bioremediation is a strategy for the removal of PAHs from the environment, which uses enzymes ormicroorganisms that have the ability to metabolize these compounds and turn them into less toxic substances. In the present study the structural characterization of the NahB protein and the biochemical and structural characterizations of NahG were done. The nahB and nahG genes were cloned and expressed in Escherichia coli RosettaTM (DE3) cells at 18° C for 16 hours, leading to expression of the enzymes in the soluble fraction. The recombinant proteins were purified by affinity and molecular exclusion chromatographies. In order to obtain folded, stable and monodisperse proteins, different protocols for cell lysis and purification were tested, besides the addition of ligands. After optimization, protein crystals of both enzymes were obtained and analyzed using a X-ray diffraction experiment. 6xHis-NahB crystal diffracted to 1.64 Å and its structure was solved by the Molecular Replacement method. The6xHis-NahB enzyme has the classic Rossman motif of nucleotide binding, comprising a central beta-sheet consisting of seven -strands and nine -helices, having three helices each side flanking the -sheet. The heavy atoms method was used to solve the 6xHis-NahG threedimensional structure. A native crystal of the 6xHis-NahG enzyme diffracted to 2.20 Å and an iodine derivative to 2.00 Å. This enzyme is characterized by the presence of three -sheets composed of eight, four and three -strands and fourteen -helices, and is structurally similarto other FAD-dependent hydroxylases. The 6xHis-NahG converts salicylic acid to catechol by descarboxylative hydroxylation and KM values of the enzyme without six histidines tag were 27.31, 1.26 and 22.74 M for the substrate salicylic acid, FAD and NADH coenzymes, respectively.
dc.publisherUniversidade Federal de Minas Gerais
dc.publisherUFMG
dc.rightsAcesso Aberto
dc.subjectBiorremediação
dc.subjectNaftaleno desidrogenase
dc.subjectSalicilato hidroxilase
dc.subjectPseudomonas
dc.subjectDifração de raios-X
dc.titleCaracterização estrutural das enzimas cis-naftaleno dihidrodioldesidrogenase e salicilato hidroxilase recombinantes de Pseudomonas putida G7 envolvidas na degradação do naftaleno
dc.typeTese de Doutorado


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