Crystallization and preliminary X-ray diffraction studies of a terminal oxygenase of carbazole 1,9a-­dioxygenase from Novosphingobium sp. KA1

The terminal oxygenase component (Oxy) of carbazole 1,9a-dioxygenase (CARDO) catalyzes dihydroxylation of the aromatic ring. The Oxy of CARDO from Novosphingobium sp. KA1 was crystallized and the crystals diffracted to a resolution of 2.1 Å.

Abstract

Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1 Å. The crystals belonged to the monoclinic space group P2₁. Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9 ų Da⁻¹ and 79.1%, respectively.

1. Introduction

Carbazole, a toxic and carcinogenic compound (Sverdrup et al., 2002 ▶; Jha & Bharti, 2002 ▶), is a component of creosote, crude oil and shale oil (Mushrush et al., 1999 ▶). In order to investigate the biodegradation of this N-heterocyclic aromatic compound in contaminated soil, many carbazole-degrading bacteria have been isolated. From analyses of the metabolism of carbazole by several bacteria, genes encoding carbazole-degradative enzymes, called the car gene cluster, have been found. Subsequent enzymatic investigation revealed the importance of carbazole 1,9a-dioxygenase (CARDO) as the initial catalyst in the carbazole-degradation pathway (Nojiri & Omori, 2007 ▶).

CARDO catalyzes regioselective and stereoselective dihydroxyl­ation of the angular (C_9a) and adjacent (C₁) C atoms of carbazole. CARDO consists of a terminal oxygenase (Oxy; encoded by the carAa gene) and the electron-transfer components ferredoxin (Fd; encoded by the carAc or fdx gene) and ferredoxin reductase (Red; encoded by the carAd or fdr gene). The electron required for the oxidation of carbazole is transferred from NAD(P)H to Oxy (Fig. 1 ▶). Novosphingobium sp. KA1 possesses the carbazole-degradative plasmid pCAR3, which contains two car gene clusters involved in carbazole assimilation and several orphan genes for electron transfer (carAaIAcI, carAaIIAcII, fdxI and fdrI/fdrII; Urata et al., 2006 ▶).

Figure 1.

Components and functions of the class IIA CARDO system. The proposed electron-transfer and dioxygenation reactions are illustrated. The cofactors of each component are shown in italics. The subscripts ‘ox’ and ‘red’ indicate oxidized and reduced states of the CARDO components, respectively.

CARDO is a member of the Rieske nonhaem iron oxygenases (ROs) that are found in various species of bacteria. ROs have been divided into five subgroups (IA, IB, IIA, IIB and III) based on the number of constituents and the nature of their redox centres (Batie et al., 1991 ▶). The CARDOs of KA1 are classified as class IIA ROs, while the well studied CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3 and Nocardioides aromaticivorans IC177 belong to classes III, III and IIB, respectively (Nojiri & Omori, 2007 ▶). In this paper, the Oxys from KA1, CA10, J3 and IC177 are abbreviated Oxy_IIA, Oxy_III, Oxy_III and Oxy_IIB, respectively. The same method of abbreviation is used for the Fds and Reds of each strain.

Several recent studies have described the structure of RO oxygen­ases such as naphthalene dioxygenase (Kauppi et al., 1998 ▶), biphenyl dioxygenase (Furusawa et al., 2004 ▶), 2-oxoquinoline 8-monooxy­genase (Martins et al., 2005 ▶) and dicamba monooxygenase (D’Ordine et al., 2009 ▶; Dumitru et al., 2009 ▶). Naphthalene dioxygenase and biphenyl dioxygenase exist as α₃β₃ heterohexamers. CARDO, 2-­oxoquinoline 8-monooxygenase and dicamba monooxygenase form α₃ homotrimers and phthalate dioxygenase is thought to be an α₃α₃ homohexamer (Tarasev et al., 2007 ▶).

To date, we have determined the crystal structures of several components of CARDO: Fd_III from P. resinovorans CA10 (Nam et al., 2005 ▶), Oxy_III from Janthinobacterium sp. J3 (Nojiri et al., 2005 ▶) and the Oxy_III–Fd_III electron-transfer complex (Ashikawa et al., 2006 ▶) and Oxy_IIB and Fd_IIB from N. aromaticivorans (Inoue et al., 2009 ▶). We have also successfully crystallized Fd_IIA and Red_IIA from Novosphingobium sp. KA1 (Umeda et al., 2008 ▶, 2010 ▶) and Red_III from Janthiobacterium sp. J3 (Ashikawa et al., 2007 ▶) and the structures of these proteins have been solved (Ashikawa et al., in preparation; Umeda et al., in preparation).

This report describes the crystallization and preliminary X-ray diffraction study of Oxy_IIA (encoded by carAaII; a trimer of 383 residues with a molecular mass of 129.3 kDa).

2. Materials and methods

2.1. Protein expression and purification

The DNA fragment containing the carAaII gene, corresponding to amino acids 1–383 of Oxy_IIA (NCBI accession No. YP_717942), was amplified from the total DNA of Novosphingobium sp. KA1 by PCR using the following oligonucleotide primers containing artificial NdeI and XhoI sites (shown in bold): 5′-AAAAAACATATGCAGACGGCAAGCGTCCCGGC-3′ and 5′-AAAAAACTCGAGTCGGACGTCCCTCGTCTGGA-3′. The PCR product was digested with NdeI and XhoI and then ligated into the corresponding sites of pET26b(+), which expresses full-length Oxy_IIA with an LEHHHHHH tag that replaces the termination codon. The integrity of the cloned constructs was confirmed by DNA sequencing and the plasmid was designated pEKA232. Escherichia coli BL21 (DE3) cells (Novagen, Wisconsin, USA; Merck, Darmstadt, Germany) transformed with pEKA232 were grown in 5 ml lysogeny medium (10 g l⁻¹ tryptone peptone, 5 g l⁻¹ yeast extract and 10 g l⁻¹ NaCl; Sambrook & Russell, 2001 ▶) supplemented with 50 µg ml⁻¹ kanamycin at 310 K. After overnight incubation, the whole culture was added to 1.5 l SB medium (24 g yeast extract, 12 g tryptone, 3.8 g KH₂PO₄, 12.5 g K₂HPO₄ and 5 ml glycerol per litre) and cultivation was carried out at 298 K and 120 rev min⁻¹. When the optical density at 600 nm reached 0.5, isopropyl d-β-1-thiogalactopyranoside was added to a final concentration of 0.5 mM. After 15 h incubation, the cells were harvested by centrifugation (5000g, 10 min), washed twice with TG buffer (20 mM Tris–HCl pH 7.5 and 10% glycerol) and resuspended in buffer A (TG buffer containing 0.5 M NaCl). Following cell lysis by sonication on ice, the lysate was clarified by centrifugation (25 000g, 60 min). All purification procedures were carried out at 277 K. The supernatant was applied onto a HiTrap Chelating HP column (GE Healthcare, Uppsala, Sweden) on an ÄKTA FPLC instrument (GE Healthcare). Oxy_IIA was eluted using a gradient of 10–200 mM imidazole in buffer A. The fraction containing Oxy_IIA was buffer-exchanged with GFC buffer (20 mM Tris–HCl pH 7.5, 0.2 M NaCl and 10% glycerol) and concentrated by ultrafiltration using a Centriprep YM-10 (Millipore, Massachusetts, USA). The concentrated solution was loaded onto a Superdex 200 prep-grade column (GE Healthcare) pre-equilibrated with GFC buffer. The eluate was buffer-exchanged with crystallization buffer (5 mM Tris–HCl pH 7.5) and concentrated as described above. The homogeneity of Oxy_IIA was confirmed by SDS–PAGE. Protein concentrations were estimated using a protein-assay kit (Bio-Rad) with BSA as the standard (Bradford, 1976 ▶).

2.2. Crystallization

Crystallization experiments were performed at 278 K using the hanging-drop vapour-diffusion method; initial trials were carried out using Crystal Screen, Crystal Screen 2, Crystal Screen Cryo, Index and Grid Screen Ammonium Sulfate (Hampton Research, California, USA). Hanging drops, each consisting of 3 µl reservoir solution and 3 µl 20 mg ml⁻¹ protein solution, were equilibrated against 600 µl reservoir solution. Oxy_IIA crystals were obtained using Crystal Screen Cryo condition No. 4 (0.075 M Tris–HCl pH 8.5, 1.5 M ammonium sulfate and 25% glycerol) after 3–5 d incubation. After several rounds of optimization, a single crystal grew after 3 d at 278 K using a protein concentration of 20 mg ml⁻¹ and a reservoir solution con­sisting of 25% glycerol, 1.5 M ammonium sulfate and 0.1 M Tris–HCl pH 8.5. Finally, the above-mentioned reservoir solution was mixed with 0.1 M calcium chloride as an additive reagent in a 4:1 ratio to set up hanging drops. The resulting solution and 20 mg ml⁻¹ protein solution were mixed in equal amounts to obtain the crystals used for X-ray diffraction data collection.

2.3. X-ray diffraction data collection

The crystals were scooped into a nylon cryoloop and directly flash-cooled in a nitrogen stream at 100 K. Diffraction experiments were conducted on beamline NW12A at the Photon Factory, Tsukuba, Japan. The data were collected at a wavelength of 1.000 Å using a Quantum 210 CCD X-ray detector (ADSC, California, USA). A complete diffraction data set consisting of 540 images with an oscillation angle of 0.5° and 5 s exposure per image was collected to a resolution of 2.1 Å. Diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶). Data-collection and processing statistics are listed in Table 1 ▶.

Table 1. Crystal parameters and data-collection statistics.

Values in parentheses are for the highest resolution shell.

Beamline	NW12A
Wavelength (Å)	1.000
Detector	ADSC Quantum Q210
Crystal-to-detector distance (mm)	164
Rotation range per image (°)	0.5
Total rotation range (°)	270
Exposure time per image (s)	5
Resolution range (Å)	50.0–2.10
Space group	P21
Unit-cell parameters (Å, °)	a = 117.1, b = 159.0, c = 167.8, α = γ = 90.0, β = 94.5
Mosaicity (°)	0.26
Total No. of reflections	2038550
No. of unique reflections	355075 (35357)
Multiplicity	5.7 (5.7)
Average I/σ(I)	31.5 (3.5)
Completeness (%)	99.9 (100)
Rmerge† (%)	7.4 (43.6)
Overall B factor from Wilson plot (Å2)	12.0

^† R _merge = , where I_i(hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all observations i of reflection hkl.

3. Results and discussion

The amount of Oxy_IIA harvested was approximately 5 mg per litre of SB culture. SDS–PAGE analysis estimated the purified Oxy_IIA to be >95% pure (Fig. 2 ▶). As with the other classes of Oxys, Oxy_IIA eluted as a trimer from gel-filtration chromatography (data not shown).

Figure 2.

SDS–PAGE of Oxy_IIA. Samples were loaded onto a 12% polyacrylamide gel in the presence of SDS. Lane M, protein markers (kDa); lane 1, cell lysate; lane 2, pooled fraction containing Oxy_IIA after affinity chromatography; lane 3, fraction containing Oxy_IIA after gel-filtration chromatography.

The crystals of Oxy_IIA grew to dimensions of 0.2 × 0.2 × 1.0 mm after 3 d and belonged to space group P2₁, with unit-cell parameters a = 117.1, b = 159.0, c = 167.8 Å, α = γ = 90, β = 94.5° (Fig. 3 ▶). Based on Matthews coefficient calculations (Matthews, 1968 ▶), between four (58.2% solvent content) and seven trimers (26.8% solvent content) could be accommodated in the asymmetric unit, with an acceptable packing density V _M in the range 1.68–2.94 ų Da⁻¹. A self-rotation function was calculated from the scaled data from 10 to 3.0 Å resolution using MOLREP (Collaborative Computational Project, Number 4, 1994 ▶; Vagin & Teplyakov, 2010 ▶) and the sections with κ = 180° and 120° showed significant peaks (Fig. 4 ▶). The peaks in the κ = 120° section were assigned to a head-to-tail homotrimeric Oxy_IIA quaternary structure and the peaks in the κ = 180° section were considered to originate from the presence of twofold noncrystallographic symmetry. These data indicated that there were two Oxy_IIA trimers in the asymmetric unit, corresponding to an unusual packing density V _M of 5.9 ų Da⁻¹ (79.1% solvent content) for the Oxy_IIA crystal. The quaternary structure of Oxy_IIA was assumed to be doughnut-shaped, with a hole at the centre of the Oxy_IIA structure. It was supposed that water molecules can enter into this hole. This is likely to be a factor in the high solvent content. A full description of the structure determination and interpretation of the structure–function relationship will be published elsewhere.

Figure 3.

An Oxy_IIA crystal. The scale bar indicates 0.3 mm.

Figure 4.

Self-rotation function map based on structure-factor amplitudes collected from a crystal of Oxy_IIA. The (a) κ = 120° and (b) κ = 180° sections are shown for a self-rotation function calculated with all data in the resolution range 10–3 Å with a radius of integration of 30 Å.