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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 Å3 Da−1 and 79.1%, respectively.
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 (C9a) 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 ).
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 OxyIIA, OxyIII, OxyIII and OxyIIB, 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: FdIII from P. resinovorans CA10 (Nam et al., 2005 ), OxyIII from Janthinobacterium sp. J3 (Nojiri et al., 2005 ) and the OxyIII–FdIII electron-transfer complex (Ashikawa et al., 2006 ) and OxyIIB and FdIIB from N. aromaticivorans (Inoue et al., 2009 ). We have also successfully crystallized FdIIA and RedIIA from Novosphingobium sp. KA1 (Umeda et al., 2008 , 2010 ) and RedIII 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 OxyIIA (encoded by carAaII; a trimer of 383 residues with a molecular mass of 129.3 kDa).
The DNA fragment containing the carAaII gene, corresponding to amino acids 1–383 of OxyIIA (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 OxyIIA 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). OxyIIA was eluted using a gradient of 10–200 mM imidazole in buffer A. The fraction containing OxyIIA 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 OxyIIA was confirmed by SDS–PAGE. Protein concentrations were estimated using a protein-assay kit (Bio-Rad) with BSA as the standard (Bradford, 1976 ).
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. OxyIIA 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.
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 .
The amount of OxyIIA harvested was approximately 5 mg per litre of SB culture. SDS–PAGE analysis estimated the purified OxyIIA to be >95% pure (Fig. 2 ). As with the other classes of Oxys, OxyIIA eluted as a trimer from gel-filtration chromatography (data not shown).
The crystals of OxyIIA 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 Å3 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 OxyIIA 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 OxyIIA trimers in the asymmetric unit, corresponding to an unusual packing density V M of 5.9 Å3 Da−1 (79.1% solvent content) for the OxyIIA crystal. The quaternary structure of OxyIIA was assumed to be doughnut-shaped, with a hole at the centre of the OxyIIA 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.
This work was supported by a Grant-in-Aid for Scientific Research (20248010 to HN) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Institute for Bioinformatics Research Development, Japan Science Technology Agency (BIRD-JST). The use of synchrotron radiation was approved by the Photon Factory Advisory Committee and KEK (High-Energy Accelerator Research Organization), Tsukuba, Japan (proposals 2004G137, 2006G171, 2007G135 and 2008G681). TU was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists.