Crystallization and preliminary crystallographic analysis of d-aspartate oxidase from porcine kidney

d-Aspartate oxidase from porcine kidney was crystallized by the sitting-drop vapour-diffusion method. The crystal diffracted to 1.80 Å resolution.

Abstract

d-Aspartate oxidase (DDO) from porcine kidney was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystal belonged to space group P2₁, with unit-cell parameters a = 79.38, b = 144.0, c = 80.46 Å, β = 101.1°, and diffracted to 1.80 Å resolution. Molecular-replacement trials using the structure of human d-amino-acid oxidase, which is 42% identical in sequence to DDO, as a search model provided a satisfactory solution.

1. Introduction  

d-Aspartate exists abundantly as an endogenous substance in the nervous and neuroendocrine systems in mammals (Hashimoto, Kumashiro et al., 1993 ▶; Hashimoto, Nikishawa et al., 1993 ▶). Recent biological evidence has demonstrated that d-aspartate is an endogenous neurotransmitter (D’Aniello et al., 2011 ▶) and is also involved in the synthesis and secretion of hormones in endocrine tissues (D’Aniello et al., 2000 ▶). Although two d-amino-acid-degrading oxidases, d-amino-acid oxidase (DAO; EC 1.4.3.3) and d-aspartate oxidase (DDO; EC 1.4.3.1), are found in mammals, endogenous d-­aspartate is only degraded by DDO. In DDO-deficient mice, a significant increase in d-aspartate content was observed selectively in tissues in which DDO is highly expressed in wild-type mice (Huang et al., 2006 ▶).

DDO requires FAD as a cofactor to catalyze the dehydrogenation of d-aspartate to iminoaspartate, which is nonenzymatically hydrolyzed to oxaloacetate and ammonia. The reduced FAD is reoxidized by O₂ to produce hydrogen peroxide. DDO only degrades acidic amino acids such as d-aspartate, N-methyl-d-aspartate and d-glutamate, whereas in mammals neutral and basic d-amino acids are substrates of DAO (Nasu et al., 1982 ▶; Negri et al., 1988 ▶).

Recently, we purified DDO from porcine kidney, cloned the cDNA and expressed the enzyme in Escherichia coli (Yamamoto et al., 2007 ▶). The purified enzyme (pDDO) was a homotetramer and showed significant substrate activition of d-aspartate at physiological concentrations of d-aspartate and O₂. Our kinetics study suggested that the oxidized and the reduced forms of pDDO show comparable affinities for d-aspartate and that the reduced enzyme–d-­aspartate complex can react with O₂ to produce a catalytically competent oxidized enzyme–d-aspartate complex.

To elucidate the mechanism of substrate activation by pDDO, crystal structure analysis of pDDO is indispensable. Although crystal structures of DAOs from various species have been reported (Mattevi et al., 1996 ▶; Umhau et al., 2000 ▶; Kawazoe et al., 2006 ▶), no crystal structures of DDO have been reported. Here, we report the crystallization and preliminary X-ray crystallographic data of pDDO.

2. Methods and results  

2.1. Protein expression, purification and crystallization  

pDDO was overexpressed using E. coli BL21 (DE3) and purified as described previously (Yamamoto et al., 2007 ▶). E. coli BL21 (DE3) cells containing a His₆-tagged porcine ddo expression plasmid were cultured at 310 K for 4–6 h after IPTG induction. After obtaining the cell extracts, the recombinant pDDO was fractionated by ammonium sulfate precipitation. Precipitated recombinant pDDO was dissolved in 20 mM sodium phosphate pH 7.4 containing 10 µM FAD, 10 mM sodium/potassium l-tartrate, 0.5 M sodium chloride and 20 mM imidazole and applied onto a HisTrap HP column (Amersham). His₆-tagged pDDO was eluted with 20 mM sodium phosphate pH 7.4 containing 10 µM FAD, 10 mM sodium/potassium l-tartrate, 0.5 M sodium chloride and 0.5 M imidazole. Fractions containing His₆-tagged pDDO were concentrated and applied onto a gel-filtration column (TSKgel G3000SW_XL, Tosoh) pre-equilibrated with 200 mM potassium phosphate pH 6.75 containing 10 µM FAD and 0.3 mM EDTA. The obtained His₆-tagged pDDO was digested with entero­kinase using an EK Max kit (Invitrogen) for 24 h at 310 K. The resultant protein solution was applied onto a gel-filtration column (TSKgel SuperSW3000, Tosoh) again using the same buffer. SDS–PAGE analysis of the purified recombinant pDDO revealed a single band of 38 kDa, indicating that recombinant pDDO was purified to near-homogeneity. The purified enzyme showed an absorption spectrum with peaks at 374 and 452 nm that were characteristic of the oxidized form of pDDO. The obtained pDDO was concentrated to 18.7 mg ml⁻¹ in 10 mM sodium phosphate buffer pH 7.4 containing 10 µM FAD and 10 mM meso-tartrate.

Initial crystallization screening was performed by the hanging-drop vapour-diffusion method using Crystal Screen and Crystal Screen II (Hampton Research, Laguna Niguel, California, USA; Jancarik & Kim, 1991 ▶) at 293 K. A hanging drop was prepared by mixing 1.0 µl each of the protein solution and the reservoir solution and was equilibrated against 500 µl reservoir solution. Although no single crystals were obtained in the initial screening, yellow spherulites appeared under two conditions: Crystal Screen condition No. 42 [20%(w/v) PEG 8000, 50 mM potassium phosphate] and Crystal Screen condition No. 36 [8%(w/v) PEG 8000, 100 mM Tris–HCl pH 8.5]. On the basis of these two conditions, the pH value (4.6–8.5) and the concentration of PEG 8000 [5–20%(w/v)] were systematically optimized, resulting in the appearance of clusters of thin yellow plate-shaped crystals in 10%(w/v) PEG 8000, 100 mM sodium acetate pH 4.6. In order to obtain single crystals, the effects of various salts were examined. Potassium dihydrogen phosphate and sodium dihydrogen phosphate were found to be effective in solving the problem. After several trials, the optimal reservoir solution was finally determined to be 5–10%(w/v) PEG 8000, 100 mM sodium dihydrogen phosphate (or 100 mM potassium dihydrogen phosphate), 100 mM sodium acetate pH 4.7 using 6.7–18.7 mg ml⁻¹ protein solution. The sitting-drop vapour-diffusion method gave larger crystals (approximately 0.5 × 0.2 × 0.02 mm) than the hanging-drop vapour-diffusion method. In addition, anaerobic crystallization in a ‘HARD’ anaerobic chamber (Hirasawa; Senda et al., 2007 ▶) reduced the amount of precipitate, resulting in larger crystals. The largest plate-shaped crystal obtained under anaerobic conditions was used for diffraction data collection (Fig. 1 ▶).

Figure 1.

A crystal of the pDDO–meso-tartrate complex.

Crystallization of the pDDO–d-aspartate complex was performed by the cocrystallization method under anaerobic conditions. pDDO concentrated to 13.7 mg ml⁻¹ was mixed with an excess amount of d-­aspartate; the molar ratio of pDDO to d-aspartate was approximately 1:20 in the solution. Plate-shaped crystals appeared in 5–10%(w/v) PEG 8000, 100 mM sodium dihydrogen phosphate (or 100 mM potassium dihydrogen phosphate), 100 mM sodium acetate pH 4.7. The crystals obtained were colourless, suggesting that the pDDO in the crystals was in the reduced form.

2.2. X-ray diffraction analysis  

Diffraction data were collected from pDDO–meso-tartrate and pDDO–d-aspartate crystals at 95 K using an ADSC CCD detector on beamline BL-17A of the Photon Factory (PF; Tsukuba, Japan; Fig. 2 ▶) and beamline NW-12A of PF-AR, respectively. Prior to data collection, the pDDO–meso-tartrate crystals were soaked in a cryoprotectant solution consisting of 20%(v/v) glycerol, 20%(w/v) PEG 8000, 100 mM sodium dihydrogen phosphate, 100 mM sodium acetate pH 4.7 for 60 s and flash-cooled in an N₂ stream. In order to maintain the reduced state of the pDDO–d-aspartate crystals, they were soaked in a cryoprotectant solution and then cooled using liquid nitrogen under anaerobic conditions.

Figure 2.

An X-ray diffraction pattern of a pDDO–meso-tartrate crystal. The diffraction data were collected on beamline BL-17A of PF using an ADSC Quantum 270 CCD detector.

The diffraction data were processed and scaled using the programs XDS and XSCALE (Kabsch, 2010 ▶). The crystals belonged to space group P2₁ (Table 1 ▶). Assuming four subunits of pDDO (37.3 kDa) per asymmetric unit of the crystal, the Matthews coefficient (V _M) was calculated to be 3.03 ų Da⁻¹, which corresponds to a solvent content of 59.4% (Matthews, 1968 ▶).

Table 1. Data-collection statistics.

Values in parentheses are for the outermost resolution shell.

	meso-Tartrate complex	D-Aspartate complex
Beamline	BL-17A, PF	NW-12A, PF-AR
Oscillation angle (°)	0.5	0.5
Crystal-to-detector distance (mm)	213.9	189.6
Exposure time (s)	10	10
Wavelength (Å)	1.0000	1.0000
Temperature (K)	95	95
Space group	P21	P21
Unit-cell parameters (Å, °)	a = 79.38, b = 144.0, c = 80.46, β = 101.1	a = 79.32, b = 143.8, c = 80.01, β = 100.9
Resolution (Å)	17.0–1.80 (1.89–1.80)	17.0–2.05 (2.15–2.05)
Observations	815711 (109097)	514738 (62167)
Unique reflections	163191 (22081)	108738 (14125)
Completeness (%)	99.7 (99.4)	98.8 (96.7)
Multiplicity	5.0 (4.9)	4.7 (4.4)
Average I/σ(I)	23.5 (4.2)	18.3 (4.0)
Rmerge (%)	0.040 (0.470)	0.055 (0.466)
Mosaicity (°)	0.139	0.325

Molecular-replacement calculations were performed with the program MOLREP (Vagin & Teplyakov, 2010 ▶) in the CCP4 program suite (Winn et al., 2011 ▶) using the structure of human d-amino-acid oxidase (PDB entry 2du8; Kawazoe et al., 2006 ▶) as a search model. The molecular-replacement calculation yielded a satisfactory solution. Packing analysis showed that there were no close contacts between the subunits. As predicted from the V _M value, the asymmetric unit contains four pDDO molecules. Crystallographic refinement of pDDO–meso-tartrate and pDDO–d-aspartate is in progress.