Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/354194
Type: Artigo
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.
Aparicio, Ricardo
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
Subject: Pseudomonas
Cinética
Country: Estados Unidos
Editor: Elsevier
Rights: Fechado
Identifier DOI: 10.1016/j.abb.2015.05.006
Address: https://www.sciencedirect.com/science/article/abs/pii/S0003986115002520
Date Issue: 2015
Appears in Collections:IQ - Artigos e Outros Documentos
IFGW - Artigos e Outros Documentos

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