Preliminary crystallographic analysis of salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Salicylate 1,2-dioxygenase, a new ring-fission dioxygenase from the naphthalenesulfonate-degrading strain P. salicylatoxidans, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring-fission mechanism, has been crystallized. The crystals obtained give diffraction data to 2.9 Å resolution which could assist in the elucidation of the catalytic mechanism of this peculiar dioxygenase.

Abstract

Salicylate 1,2-dioxygenase, a new ring-fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring-fission mechanism, has been crystallized. Diffraction-quality crystals of salicylate 1,2-dioxygenase were obtained using the sitting-drop vapour-diffusion method at 277 K from a solution containing 10%(w/v) ethanol, 6%(w/v) PEG 400, 0.1 M sodium acetate pH 4.6. Crystals belong to the primitive tetragonal space group P4₃2₁2 or P4₁2₁2, with unit-cell parameters a = 133.3, c = 191.51 Å. A complete data set at 100 K extending to a maximum resolution of 2.9 Å was collected at a wavelength of 0.8423 Å. Molecular replacement using the coordinates of known extradiol dioxygenases structures as a model has so far failed to provide a solution for salicylate 1,2-dioxygenase. Attempts are currently being made to solve the structure of the enzyme by MAD experiments using the anomalous signal of the catalytic Fe^II ions. The salicylate 1,2-dioxygenase structural model will assist in the elucidation of the catalytic mechanism of this ring-fission dioxygenase from P. salicylatoxidans, which differs markedly from the known gentisate 1,2-­dioxygenases or 1-hydroxy-2-naphthoate dioxygenases because of its unique ability to oxidatively cleave salicylate, gentisate and 1-hydroxy-2-naphthoate with high catalytic efficiency.

1. Introduction

The oxygenolytic cleavage of the aromatic nucleus by bacteria as a general rule demands the presence of two hydroxyl groups attached to the aromatic ring (Bugg, 2003 ▶; Costas et al., 2004 ▶). Only a few instances have been described in which monohydroxylated aromatic compounds have been cleaved by ring-fission dioxygenases (Davis et al., 1999 ▶; Harpel & Lipscomb, 1990b ▶). Recently, a new ring-fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring-fission mechanism, has been described (Fig. 1 ▶; Hintner et al., 2001 ▶, 2004 ▶).

Figure 1.

Salicylate dioxygenase-catalyzed oxidative cleavage of (substituted) salicylate(s): R ₁, R ₂, R ₃ = H, NH₂, OH, CH₃, F, Cl, Br, I.

The salicylate dioxygenase activity of P. salicylatoxidans BN12 is unique among the currently known ring-fission dioxygenases in that the enzyme is able to cleave various substituted salicylates that carry only a single hydroxy group and that are not activated for a ring-fission reaction by additional electron-donating substituents. Previous biochemical characterization of the salicylate dioxygenase activity from P. salicylatoxidans BN12 demonstrated that in addition to salicylate, the enzyme also converts gentisate, 5-aminosalicylate and 1-hydroxy-2-naphthoate, 3-amino- and 3- and 4-hydroxysalicylate, 5-­fluorosalicylate, 3-, 4- and 5-chlorosalicylate, 3-, 4- and 5-bromosalicylate, 3-, 4- and 5-methylsalicylate and 3,5-dichlorosalicylate (see Fig. 1 ▶).

Sequence alignments and gel-filtration experiments suggested salicylate dioxygenase to be structurally similar to gentisate 1,2-dioxygenase from different microorganisms, such as Comamonas testosteroni, C. acidovorans, Haloferax sp., Klebsiella pneumoniae, Moraxella osloensis OA3, Pseudomonas alcaligenes and Sphingomonas sp. strain RW5 (Crawford et al., 1975 ▶; Feng et al., 1999 ▶; Fu & Oriel, 1998 ▶; Harpel & Lipscomb, 1990a ▶; Hintner et al., 2001 ▶, 2004 ▶; Luo et al., 2006 ▶; Suarez et al., 1996 ▶; Werwath et al., 1998 ▶; Zhou et al., 2001 ▶).

This was indicated by the size of the subunits (about 40 kDa), the structure of the holoenzyme (tetramer) and the dependence of the enzyme on Fe²⁺ ions (one iron per monomer). Nevertheless, it became evident that the ring-fission dioxygenase from P. salicylatoxidans was clearly different from the presently known gentisate 1,2-­dioxygenases or 1-hydroxy-2-naphthoate dioxygenases because of its unique ability to oxidatively cleave salicylate and also its ability to cleave gentisate and 1-hydroxy-2-naphthoate with high catalytic efficiencies (Hintner et al., 2001 ▶, 2004 ▶).

The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The deduced amino-acid sequence encoded a protein with a molecular weight of 41 176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 (GenBank accession No. AAD49427) and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7 (GenBank accession No. BAA31235) (Hintner et al., 2004 ▶).

In order to allow a more detailed analysis of the relationship between the mechanistic capabilities of this particular ring-fission dioxygenase and its structural features, this enzyme was crystallized and X-ray diffraction data were collected.

2. Experimental procedures

2.1. Protein purification

Salicylate 1,2-dioxygenase from P. salicylatoxidans strain BN12 DSM 6986 was heterologously expressed in E. coli JM109 (pJPH100exN) as reported by Hintner et al. (2004 ▶). The His-tagged enzyme variant was purified, tested for activity and analyzed for purity as previously described (Hintner et al., 2004 ▶).

2.2. Crystallization

The enzyme was concentrated to 19 mg ml⁻¹ in 20 mM Tris–HCl pH 8, 100 mM sodium chloride using a Centricon ultraconcentrator (10 kDa molecular-weight cutoff, Amicon).

Crystallization experiments were performed using the sitting-drop vapour-diffusion method and 96-well plates (CrystalQuick, Greiner Bio-One, Germany).

Initial crystallization trials were performed using Structure Screens I and II from Molecular Dimensions Ltd and JBScreen Classic from Jena Bioscience at 277 K. Condition C2 of the JBScreen Classic 8 [12%(w/v) ethanol, 4%(w/v) PEG 400, 100 mM sodium acetate pH 4.6] was chosen as the most promising and was optimized by modifying the concentration of the different components and the pH of the buffer.

Diffraction-quality crystals were obtained at 277 K from a solution containing 8–12%(w/v) ethanol, 6%(w/v) PEG 400, 0.1 M sodium acetate pH 4.6. Drops were prepared using 1 µl protein solution mixed with 1 µl reservoir solution and were equilibrated against 100 µl precipitant solution.

2.3. X-ray data collection

A complete data set extending to a maximum resolution of 2.9 Å was collected at 100 K on EMBL beamline BW7B, Hamburg, Germany. After adding 30% glycerol to the mother liquor as a cryoprotectant, data were collected using a MAR 345 image-plate detector and a wavelength of 0.8423 Å (Fig. 2 ▶). The crystals showed no significant decay upon exposure.

Figure 2.

Area-detector frame showing the diffraction spots for crystals of salicylate 1,2-dioxygenase from P. salicylatoxidans.

3. Results

Under the optimal conditions (see §2), crystals of salicylate 1,2-dioxygenase from P. salicylatoxidans grow within one week at 277 K using the sitting-drop vapour-diffusion method to approximate dimensions of 0.2 × 0.2 × 0.2 mm (Fig. 3 ▶).

Figure 3.

Microphotography of salicylate 1,2-dioxygenase from P. salicylatoxidans crystals obtained by the sitting-drop vapour-diffusion method.

Crystals belong to the primitive tetragonal space group P4₃2₁2 or P4₁2₁2, with unit-cell parameters a = 133.3, c = 191.51 Å. Assuming one tetramer per asymmetric unit, the solvent content is 47% of the unit cell (Matthews coefficient V _M = 2.3 ų Da⁻¹; Matthews, 1968 ▶).

Data processing with MOSFLM and SCALA gave 38 218 unique reflections, an R _sym of 12.1% and an overall completeness of 99.0%. Statistics of the data collection and processing are reported in Table 1 ▶.

Table 1. Crystal parameters and data-collection statistics.

Beamline	BW7B, DESY, Hamburg
Space group	P43212 or P41212
Unit-cell parameters
a (Å)	133.02
c (Å)	190.75
Asymmetric unit content	1 molecule
VM (Å3 Da−1)	2.3
Solvent content (%)	47
Wavelength (Å)	0.8423
Resolution limits (Å)	20–2.9 (3.11–2.9)
Total reflections measured	749226
Unique reflections	38218
Rsym†	0.12 (0.47)
Multiplicity	4.8 (4.9)
Completeness (%)	99.0 (99.6)
I/σ(I)	4.4 (1.7)

^† R _sym = , where I_i is an individual intensity measurement and 〈I〉 is the average intensity for this reflection with summation over all data.

Salicylate 1,2-dioxygenase from P. salicylatoxidans shows sequence homology to several gentisate 1,2-dioxygenases, the structure of which is still unknown. We have attempted to carry out molecular replacement with the program MOLREP (Vagin & Teplyakov, 1997 ▶) using models of known extradiol dioxygenase structures, among them the quercetin 2,3-dioxygenase from Aspergillus japonicus, which shares the highest sequence homology with salicylate 1,2-dioxygenase from P. salicylatoxidans (PDB code 1gqg; 15.1% sequence identity; Steiner et al., 2002 ▶). Furthermore, a homology search against protein sequences from the Protein Data Bank was carried out using FFAS (http://ffas.ljcrf.edu) and the structures with the highest sequence identity were used as initial models for molecular-replacement calculations. All these attempts were unsuccessful in finding a solution for salicylate 1,2-dioxygenase from P. salicylatoxidans.

Since salicylate 1,2-dioxygenase from P. salicylatoxidans contains Fe^II ions, it may have sufficient anomalous signal for the multiple anomalous dispersion (MAD) method. Attempts will be made to solve the structure of the enzyme by a MAD experiment using the anomalous signal of the catalytic Fe^II.