Expression, purification and preliminary crystallographic studies of NahF, a salicylaldehyde dehydrogenase from Pseudomonas putida G7 involved in naphthalene degradation

Abstract

Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.

Figure 1

Naphthalene-degradation upper pathway. The upper pathway is composed of NahAaAbAcAd (naphthalene dioxygenase complex; NDO), cis-naphthalene dihydrodiol dehydrogenase (NahB), 1,2-dihydroxynaphthalene dioxygenase (NahC), 2-hydroxy-2H-chromene-2-carboxylate isomerase (NahD), trans-o-hydroxybenzyl­idenepyruvic hydratase-aldolase (NahE) and salicylaldehyde dehydrogenase (NahF). The main substrates and products for these enzymes are also depicted. (1) Naphthalene, (2) cis-1,2-dihydroxy-1,2-dihydronaphthalene, (3) 1,2-dihydroxy­naphthalene, (4) 2-hydroxy-2H-chromene-2-carboxylic acid, (5) trans-o-hydroxybenzylidenepyruvic acid, (6) salicylaldehyde, (7) pyruvate and (8) salicylic acid (adapted from Peng et al., 2008 ▶).

Figure 2

Typical size-exclusion chromatography chromatogram obtained with recombinant NahF. The elution of recombinant NahF in peak 3 (the main peak) corresponds to the profile of a 103 kDa protein and is in good agreement with a dimeric form of 6×His-NahF (108 kDa). The elution volume (V _e) and molecular mass (MM) associated with each peak (1, 2 and 3) are shown.

Figure 3

Flower-shaped crystals of recombinant NahF. Crystals were obtained using 1.5 M ammonium sulfate, 5%(v/v) 2-propanol, 5 mM NAD⁺ in 100 mM sodium acetate–acetic acid buffer pH 5.0.

Figure 4

A diffraction image from a broken piece of a flower-shaped 6×His-NahF crystal. Diffraction data were collected to 2.42 Å resolution. The diffraction pattern background around the centre of the image was altered to allow a better visualization of the reflections. Resolution shells are shown (in Å) close to their respective circles.