Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.
Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.
复制标题
恶臭假单胞菌 G7 重组 2-羟基粘康酸半醛脱氢酶的结构和动力学表征。
DOI：
10.1016/j.abb.2015.05.006
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发表时间：
2015
影响因子：
3.9
通讯作者：
Nagem,RonaldoAlvesPinto
中科院分区：
文献类型：
--
作者：
Araújo,SimaraSemíramisde;Neves,CíntiaMaraLeal;Guimarães,SamuelLeite;Whitman,ChristianP;JohnsonJr,WilliamH;Aparicio,Ricardo;Nagem,RonaldoAlvesPinto
The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway inPseudomonas putidaG7 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. ThenahIgene was subcloned into a T7 expression vector and the enzyme was overexpressed inEscherichia coliArcticExpress 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 μM) andkcat(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.
