Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular-position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 Å resolution and belong to space group C2.

The carAa gene (accession No. BAE79498) was PCR-amplified from plasmid pB177103 (Inoue et al., 2006 ) using primers 5′-TCTAGAGTAAGGAGGTGTTCATATGAGCACCTCTCAGGAAAT-3′ and 5′-ACTAGTAAGCTTTCAGTGGTGGTGGTGGTGGTGCGACATTTCCACTCGGGC-3′. The PCR product was ligated into the pT7Blue T-vector (Novagen). The nucleotide sequence of the insert was checked with the original sequence. The plasmid was digested with NdeI and HindIII. A 1.2 kbp NdeI–HindIII fragment was inserted into overexpression vector pET-26b(+) (Novagen; designated pE177503). pE177503 contains the genes for the full-length CARDO-O subunit with a 6×His tag that replaced the termination codon (ht-CARDO-O). The transformed Escherichia coli BL21 (DE3) (Novagen) cells were grown at 303 K on SB medium (Nam et al., 2002 ) supplemented with 0.5 mM IPTG. After 12 h incubation, the cells were harvested by centrifugation at 5000g for 10 min, washed twice with TG buffer (Nam et al., 2002 ) and resuspended in buffer A (20 mM Tris–HCl pH 7.5 containing 0.5 M NaCl and 10% glycerol). The crude cell extract was prepared by sonication and centrifugation at 25 000g for 2 h and was applied onto a HiTrap Chelating HP column (GE Healthcare) equipped with an ÄKTA FPLC instrument (GE Healthcare) according to the manufacturer’s recommendations. ht-CARDO-O was eluted with buffer B (buffer A containing 300 mM imidazole). The fractions containing ht-CARDO-O were pooled and concentrated by ultrafiltration using Centriprep YM-10 (Millipore). The resultant preparation was further purified by gel-filtration chromatography using a Superdex 200 prep-grade (GE Healthcare) column and GFC buffer (Nam et al., 2002 ). During purification using gel-filtration chromatography, the molecular weight of the putative α₃ trimer of ht-CARDO-O was estimated as 124 kDa. As the molecular weight of the monomer corresponded to approximately 45 kDa on SDS–PAGE (data not shown), this suggested that ht-CARDO-O indeed forms an α₃ assembly. Prior to crystallization, the purified ht-CARDO-O was confirmed to retain its angular dioxygenation activity for carbazole when coupled with the electron-transfer proteins CARDO-F and CARDO-R from N. aromaticivorans IC177 and NADH (data not shown; Fig. 1 ). These data indicated that CARDO-O from N. aromaticivorans IC177 is active as a trimer similar in nature to that of CARDO from P. resinovorans CA10 (Nam et al., 2002 ). Protein concentrations were estimated using a protein-assay kit (Bio-Rad; Bradford, 1976 ) with BSA as a standard. For crystallization experiments, a solution of the protein in 5 mM Tris–HCl pH 7.5 with CARDO-O concentration in the range 5–­30 mg ml⁻¹ was used.

Crystallization was performed using the hanging-drop vapour-diffusion method at 293 K. Drops containing 2 µl protein solution and 2 µl mother liquor were equilibrated against 800 µl reservoir solution. The initial crystallization conditions were screened using Crystal Screens I and II, Crystal Screen Cryo and Index (Hampton Research). Several crystals were obtained using Crystal Screen Cryo condition No. 50 [12%(w/v) PEG 8000, 0.4 M lithium sulfate and 20%(v/v) glycerol] in the reservoir and protein solution at a concentration of 30 mg ml⁻¹. The crystals appeared within 3 d and grew to approximate dimensions of 0.2 × 0.2 × 0.02 mm (Fig. 2 ). Attempts to optimize this condition by changing the pH, precipitant concentration and temperature did not improve the quality of the crystals.

The crystals were directly flash-cooled in a nitrogen stream at 100 K. Diffraction experiments were conducted at beamline AR-NW12, Photon Factory, Tsukuba, Japan. Diffraction data were collected using a wavelength of 1.0 Å with a Quantum 210 CCD X-ray detector (ADSC). The diffraction data were collected using a single crystal in 0.5° oscillation steps over a range of 360° with a 5 s exposure per frame. The data sets were integrated and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997 ). A data set was collected to 2.3 Å resolution. The data-collection and processing statistics are summarized in Table 1 . The space group of the crystal was determined to be C2, with unit-cell parameters a = 280.00, b = 161.66, c = 194.66 Å, β = 118.65°. Initial analysis of the crystal solvent content using the Matthews coefficient (Matthews, 1968 ) suggested that the asymmetric unit contains five trimers (57.4% solvent content) or six trimers (48.9% solvent content), with an acceptable packing density V _M of 2.88 or 2.40 ų Da⁻¹, respectively. The crystal structure solution was attempted using the molecular-replacement method with the structure of ht-CARDO-O from Sphingomonas sp. KA1 (49% amino-acid sequence identity; Katsuki et al., structural data unpublished) as a search model. A full description of the structure determination will be published elsewhere.