title: 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.
abstract: The 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 Kmvalues of 6 and 350 μM, and a kcatvalue 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.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Débora Maria Abrantes
authors.initials: DMA
authors.lastname: Costa
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Mariana Amalia Figueiredo
authors.initials: MAF
authors.lastname: Costa
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Samuel Leite
authors.initials: SL
authors.lastname: Guimarães
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Juliana Barbosa
authors.initials: JB
authors.lastname: Coitinho
authors.affiliation: Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Stefanya Velásquez
authors.initials: SV
authors.lastname: Gómez
authors.affiliation: Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil.
authors.forename: Tiago Antônio da Silva
authors.initials: TADS
authors.lastname: Brandão
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, MG, CEP 31270-901, Brazil. Electronic address: nagem@icb.ufmg.br.
authors.forename: Ronaldo Alves Pinto
authors.initials: RAP
authors.lastname: Nagem
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000072417:Protein Domains; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C020054:cis-1,2-dihydrobenzene-1,2-diol dehydrogenase
keywords: NahB; Naphthalene degradation; Protein stability; Pseudomonas putida G7; Structural characterization; cis-dihydrodiol dehydrogenase