A combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme

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dc.creatorDébora Maria Abrantes Costa
dc.creatorMariana Amalia Figueiredo Costa
dc.creatorSamuel Leite Guimarães
dc.creatorJuliana Barbosa Coitinho
dc.creatorStefanya Velásquez Gómez
dc.creatorTiago Antônio da Silva Brandão
dc.creatorRonaldo Alves Pinto Nagem
dc.date.accessioned2023-09-29T11:53:22Z
dc.date.accessioned2025-09-09T01:19:59Z
dc.date.available2023-09-29T11:53:22Z
dc.date.issued2017
dc.description.sponsorshipCNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
dc.description.sponsorshipFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
dc.description.sponsorshipOutra Agência
dc.identifier.doihttps://doi.org/10.1016/j.pep.2017.01.005
dc.identifier.issn1096-0279
dc.identifier.urihttps://hdl.handle.net/1843/59024
dc.languageeng
dc.publisherUniversidade Federal de Minas Gerais
dc.relation.ispartofProtein Expression and Purification
dc.rightsAcesso Restrito
dc.subjectPseudomonas
dc.subjectAnálise enzimática
dc.subjectBiorremediação
dc.subjectBioquímica
dc.subject.otherNaphthalene degradation
dc.subject.otherCis-dihydrodiol dehydrogenase NahB
dc.subject.otherPseudomonas putida G7
dc.subject.otherProtein stability
dc.subject.otherStructural characterization
dc.titleA combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme
dc.typeArtigo de periódico
local.citation.epage59
local.citation.spage50
local.citation.volume132
local.description.resumoThe second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 μM, and a kcat value of 8.3 s−1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.
local.identifier.orcidhttps://orcid.org/0000-0001-7628-1871
local.identifier.orcidhttp://orcid.org/0000-0002-7316-604X
local.identifier.orcidhttps://orcid.org/0000-0002-3699-3927
local.identifier.orcidhttps://orcid.org/0000-0002-5892-050X
local.identifier.orcidhttps://orcid.org/0000-0002-7783-3014
local.identifier.orcidhttps://orcid.org/0000-0001-7219-1625
local.publisher.countryBrasil
local.publisher.departmentICB - DEPARTAMENTO DE BIOQUÍMICA E IMUNOLOGIA
local.publisher.departmentICX - DEPARTAMENTO DE QUÍMICA
local.publisher.initialsUFMG
local.url.externahttps://www.sciencedirect.com/science/article/pii/S1046592816303837

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