Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7

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.

Keywords: 2-Hydroxymuconate semialdehyde dehydrogenase; Kinetics; Naphthalene degradation; Pseudomonas putida G7; Structure.

Figure 1

Naphthalene-degradation lower pathway (from salicylate to pyruvate and acetyl-CoA). This pathway is composed of the enzymes salicylate hydroxylase (NahG), catechol 2,3-dioxygenase (NahH), 2-hydroxymuconate semialdehyde dehydrogenase (NahI), 4-oxalocrotonate tautomerase (NahJ), 4-oxalocrotonate decarboxylase (NahK), 2-hydroxymuconate semialdehyde hydrolase (NahN), 2-oxopent-4-enoate hydratase (NahL), 4-hydroxy-2-oxopentanoate aldolase (NahM), and acetaldehyde dehydrogenase (NahO). The main substrates and products for these enzymes are also depicted. The metabolites are: naphthalene (1), salicylate (2), catechol (3), 2-hydroxymuconate semialdehyde (4), 2-hydroxymuconate (5), 2-oxo-3-hexenedioate (6), 2-hydroxy-2,4-pentadienoate (7), 4-hydroxy-2-oxopentanoate (8), acetaldehyde (9), pyruvate (10), and acetyl-CoA (11).

Figure 2

Coomassie blue-stained 15% SDS–PAGE analysis of 6xHis-NahI expression in E. coli ArcticExpress (DE3) and purification. Lane MW: Protein molecular weight marker; Lane 0h: crude bacterial extract before IPTG induction; Lane 24h: crude bacterial extract after 24h of induction with 1.0 mM IPTG; Lanes S and I: soluble and insoluble fractions, respectively, after cell lysis. Lanes AC and SEC: purified fractions of 6xHis-NahI after Ni²⁺-affinity and size-exclusion chromatography.

Figure 3

A. CD spectra of GH-NahI at 20 ºC and 95 ºC, in the absence and presence of NAD⁺, in 50 mM sodium phosphate buffer pH 7.4 and 50 mM NaCl. The protein concentration in both solutions is 0.3 mg/mL and the cell path lengths are 0.5 mm. B. CD melting curves recorded at 222 nm for GH-NahI and GH-NahI/NAD⁺ between 20 ºC and 95 ºC.

Figure 4

A. Size-exclusion chromatography curves for GH-NahI in the absence (dashed line, Ve = 79.3 mL / grey solid line, Ve = 76.7 mL) and presence of NAD⁺ (dotted line, Ve = 77.8 mL, black solid line / Ve = 76.7 mL). B. Calibration curve generated using molecular weight standards.

Figure 5

SAXS data and overall parameters. A. Experimental Small-Angle X-ray Scattering curve (open circles) of GH-NahI in solution, with the GNOM [33] fitting (thicker line in grey) obtained during the computation of the pair-distance distribution function, P(r). The Guinier region and the corresponding linear fitting are shown in the inset. Theoretical SAXS curves for modeled NahI dimer (thin line in black) and tetramer (thicker line in black) were calculated with FoXS program (REF1 - doi: 10.1016/j.bpj.2013.07.020 , REF2 - doi:10.1093/nar/gkq461) and fitted to SAXS experimental data. B. Pair-distance distribution function derived from the experimental curve. The bell-shaped profile, with a centered maximum, is typical of a globular scattering particle in solution. C. Kratky plot calculated from the experimental data; typical of compact proteins, with well-folded domains. A few negative intensity points resulting from buffer subtraction in the noisy region, at higher scattering angles, were deleted previously to FoXS calculations and for figures composition. Plots were done with GNUPLOT (http://www.gnuplot.info) and edited with GIMP (http://www.gimp.org) under Slackware Linux (http://www.slackware.com).

Figure 6

Superposition of the experimental envelope (shown as a surface) onto the predicted NahI tetramer, represented as a cartoon with monomer chains shown in different colors. The middle and bottom views are rotated clockwise by 90° around the x- and y-axes, respectively. The axes of the coordinate system of the page were chosen to make evident the striking coincidence of the envelope symmetry, with the nearly perfect 222 point-group symmetry of the tetramer. Drawings were prepared with PyMOL and edited using GIMP (http://www.gimp.org) under Slackware Linux (http://www.slackware.com).

Figure 7

A: Kinetic activity of the recombinant GH-NahI/NAD⁺ complex with 2-hydroxymuconate semialdehyde. A plot of the initial velocities [v_o] of the enzyme-catalyzed reaction versus the concentration of 2-hydroxymuconate semialdehyde shows that the enzyme follows Michaelis-Menten kinetics. B and C: Partial amino acid and nucleotide sequence alignment between NahI and XylG. A notable difference extending over 21 consecutive amino acid residues (Ala-87 to Ala-109) may be responsible for the different kinetic behavior observed for XylG (B). An insertion and a deletion mutation flanking this different fragment might have produced a reading frameshift (C). Predicted NahI monomer showing that the fragment (Ala-87 to Ala-109 colored in grey) stands in the vicinity of the predicted catalytic site (black).