title: Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.
abstract: The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s(-1)) values were determined at presumed NAD(+) saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil.
authors.forename: Simara Semíramis de
authors.initials: SS
authors.lastname: Araújo
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil.
authors.forename: Cíntia Mara Leal
authors.initials: CM
authors.lastname: Neves
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil.
authors.forename: Samuel Leite
authors.initials: SL
authors.lastname: Guimarães
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, TX 78712-1071, USA.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, TX 78712-1071, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Johnson
authors.affiliation: Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, CP 6154, Campinas, SP 13083-970, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Aparicio
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: nagem@icb.ufmg.br.
authors.forename: Ronaldo Alves Pinto
authors.initials: RA
authors.lastname: Nagem
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry*; D009281:Naphthalenes / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins; D013379:Substrate Specificity
chemical_list: D009281:Naphthalenes; D011994:Recombinant Proteins; D009243:NAD; C031721:naphthalene; D000445:Aldehyde Oxidoreductases; C023320:2-hydroxymuconic semialdehyde dehydrogenase
keywords: 2-Hydroxymuconate semialdehyde dehydrogenase; Kinetics; Naphthalene degradation; Pseudomonas putida G7; Structure