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Type: Artigo de periódico
Title: Structural And Kinetic Characterization Of Recombinant 2-hydroxymuconate Semialdehyde Dehydrogenase From Pseudomonas Putida G7.
Author: Araújo, Simara Semíramis de
Neves, Cíntia Mara Leal
Guimarães, Samuel Leite
Whitman, Christian P
Johnson, William H
Aparicio, Ricardo
Nagem, Ronaldo Alves Pinto
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.
Subject: 2-hydroxymuconate Semialdehyde Dehydrogenase
Naphthalene Degradation
Pseudomonas Putida G7
Citation: Archives Of Biochemistry And Biophysics. v. 579, p. 8-17, 2015-Aug.
Rights: embargo
Identifier DOI: 10.1016/
Date Issue: 2015
Appears in Collections:Unicamp - Artigos e Outros Documentos

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