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Arch Biochem Biophys ; 579: 8-17, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26032336

RESUMO

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.


Assuntos
Aldeído Oxirredutases/química , Aldeído Oxirredutases/ultraestrutura , NAD/química , Naftalenos/química , Pseudomonas putida/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Pseudomonas putida/genética , Proteínas Recombinantes , Especificidade por Substrato
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