Please use this identifier to cite or link to this item:
|Title:||Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7|
|Author:||de Araujo, Simara Semiramis|
Leal Neves, Cintia Mara
Guimaraes, Samuel Leite
Whitman, Christian P.
Johnson Junior, William H.
Pinto Nagem, Ronaldo Alves
|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 +/- 0.3 mu M) and kcat (0.9 s(-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|
|Appears in Collections:||IQ - Artigos e Outros Documentos|
IFGW - Artigos e Outros Documentos
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.