Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver

An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was successfully isolated and crystallized.

Abstract

An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroid­ectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.26, b = 81.32, c = 77.27 Å, β = 119.43°, and diffracted to 1.86 Å resolution on beamline NE3A at the Photon Factory. The overall R _merge was 5.4% and the data completeness was 99.4%.

1. Introduction  

Carbonyl reductase (CR; EC 1.1.1.184) is an NAD(P)H-dependent oxidoreductase that catalyses the reduction of a variety of quinones and other carbonyl compounds to the corresponding quinol and alcohol products. Although multiple forms of the enzyme differing in size and charge have been isolated from various sources (Wermuth, 1985 ▶), all CRs belong to the short-chain dehydrogenase/reductase (SDR) family, and the two signature sequences of the SDR family, an S–YXXXK motif in which the conserved tyrosine plays a key role in catalysis and a GXXXGXG motif which is located near the cofactor-binding pocket, are strictly conserved. Based on its substrate specificity, CR is thought to function in the metabolism of endogenous carbonyl compounds, such as aliphatic aldehydes and ketones derived from lipid peroxidation, as well as in the metabolism of xenobiotics (Nakanishi et al., 1995 ▶).

Shibata et al. (2003 ▶) have demonstrated that a 29 kDa protein with unknown function is specifically expressed in thyroid­ectomized (Tx) chicken fatty liver. On the basis of genome information on the chicken (Gallus gallus), we recently identified the gene (NCBI XP_414028) encoding this protein. From comparison of the amino-acid sequence of this protein with those of homologues for which crystal structures have been determined to date, we found that the protein exhibits the highest identity (36%) to a CR from the fruit fly Drosophila melanogaster (PDB entry 1sny; Sgraja et al., 2004 ▶) and contains the two consensus sequences (S–YXXXK and GXXXGXG) of the SDR family. Moreover, we succeeded in expressing the gene in Escherichia coli and confirmed that the gene product exhibits CR activity. Since the enzyme shows rather low sequence identities to CRs from human (33%; Tanaka et al., 2005 ▶), mouse (30%; Tanaka et al., 1996 ▶) and pig (27%; Tanaka et al., 2008 ▶), it is of interest to examine structural differences among insect, chicken and mammalian CRs. The physiological function of the CR in Tx chicken fatty liver is currently unknown. The enzyme probably contributes to the metabolism of accumulated ketone bodies derived from fatty acids. Structural analysis of the enzyme may provide important information about its physiological substrate and function. In this paper, we describe the expression, purification, crystallization and preliminary X-ray analysis of an NAD(P)H-dependent CR which is specifically expressed in the fatty liver of thyroidectomized chicken.

2. Experimental procedures and results  

2.1. Gene cloning and expression, and purification of the gene product  

The gene encoding the Tx chicken fatty liver CR was amplified using the primers presented in Supplementary Table S11 as described previously (Dillon & Rosen, 1993 ▶). The nucleotide sequence of the gene is shown in Supplementary Table S2. The amplified 0.8 kbp fragment was digested with NdeI and EcoRI, and ligated with the expression vector pET15b (Novagen, Madison, Wisconsin, USA) to generate pETCR. E. coli strain BL21 (DE3) CodonPlus RIPL (Stratagene, La Jolla, California, USA) was then transformed with the vector and the transformants were cultivated at 310 K in 200 ml Luria–Bertani medium containing 50 µg ml⁻¹ ampicillin until the optical density at 600 nm reached 0.6. Expression was then induced by adding 1.0 mM isopropyl β-d-1-thiogalacto­pyranoside (IPTG) to the medium and cultivation was continued for an additional 3 h at 310 K. E. coli cells harvested from the 200 ml culture (about 1.3 g wet weight) were used as the starting material for purification of CR. To prepare the crude extract, the cells were washed twice and suspended in 10 ml 10 mM Tris–HCl buffer pH 8.0. The cells were then disrupted by sonication and centrifuged at 20 000g for 10 min. The CR activity in the crude extract was assayed using 4-benzoylpyridine as a substrate, as described previously (Nishinaka et al., 1992 ▶).

For purification of the Tx chicken fatty liver CR, the crude extract was applied onto a Co²⁺-charged Talon resin column (10 × 40 mm; Clontech, Palo Alto, California, USA) equilibrated with 10 mM Tris–­HCl buffer pH 8.0 containing 500 mM NaCl. After washing the column with the same buffer, the enzyme was eluted using the same buffer supplemented with 300 mM imidazole. After elution, the active fractions were pooled and used as the purified enzyme preparation. The enzyme could readily be purified from the crude cell extract in one simple step; about 6 mg purified enzyme was obtained from a 200 ml E. coli culture. In addition to 4-benzoyl­pyridine, the enzyme also catalysed the reduction of 9,10-phenanthrenequinone and menadione (the relative activities were 488% and 148%, respectively) as model substrates for CR in the presence of NADPH as a coenzyme (electron donor). In addition, NADPH was a more preferable coenzyme than NADH for CR; the reaction rate with NADH (0.06 mM) was only 14% of that with NADPH (0.06 mM) when 9,10-phenanthrenequinone was used as the substrate. The highest reaction rate (5.0 µmol min⁻¹ mg⁻¹) was obtained for the reduction of 9,10-phenanthrenequinone in the presence of NADPH. Tx chicken fatty liver CR showed typical Michaelis–Menten kinetics; the K _m values for NADPH, 9,10-phenanthrenequinone, 4-benzoylpyridine and menadione were 0.017, 0.002, 8.2 and 3.5 mM, respectively.

2.2. Molecular-mass determination  

The molecular mass of the recombinant enzyme was determined using a TSKgel G3000SW_XL column (7.8 × 300 mm; Tosoh, Japan) with 10 mM potassium phosphate buffer pH 7.0 containing 150 mM NaCl as the elution buffer. A gel-filtration marker kit (Sigma–Aldrich, St Louis, Missouri, USA) was used as molecular-mass standards. The subunit molecular mass was determined by SDS–PAGE using eight marker proteins (7–175 kDa; New England Biolabs Inc.). The native and subunit molecular masses of the enzyme were determined to be about 28 and 26 kDa, respectively, suggesting that the enzyme is a monomer in solution (Fig. 1 ▶).

Figure 1.

Molecular-mass determination of Tx chicken fatty liver CR. (a) SDS–PAGE of the purified enzyme: left lane, marker proteins (labelled in kDa); right lane, purified Tx chicken fatty liver CR. (b) Molecular mass of the native enzyme as determined by gel filtration.

2.3. Crystallization  

The purified enzyme was dialysed against 10 mM Tris–HCl buffer pH 8.0 containing 200 mM NaCl and 5 mM β-mercaptoethanol and concentrated to 8 mg ml⁻¹ for crystallization trials. Initial screening was carried out with Wizard I and II and Cryo I and II (Emerald BioSystems, USA) at 293 K using the sitting-drop vapour-diffusion method, in which 1 µl drops of protein solution containing 1 mM NADP⁺ were mixed with an equal volume of reservoir solution and equilibrated against 0.1 ml reservoir solution using Compact Clover Crystallization Plates (Emerald BioSystems). The Tx chicken fatty liver CR crystals were grown using reagent No. 18 [40% polyethylene glycol (PEG) 300, 100 mM phosphate–citrate pH 4.2] of Cryo II. Diffraction-quality crystals (maximum dimensions of 0.6 × 0.1 × 0.1 mm; Fig. 2 ▶) were obtained within two weeks.

Figure 2.

Monoclinic crystal of Tx chicken fatty liver CR. The maximum dimensions of the crystal are 0.6 × 0.1 × 0.1 mm.

2.4. Data collection and preliminary X-ray analysis  

The Tx chicken fatty liver CR crystal was flash-cooled in liquid nitrogen at 100 K. The crystal was cryoprotected with a solution consisting of 42% PEG 300, 100 mM phosphate–citrate pH 4.2. Diffraction data were collected to 1.86 Å resolution using monochromated radiation of wavelength 1.0 Å and an ADSC CCD detector system on the NE3A beamline at the Photon Factory, Tsukuba, Japan. The oscillation angle per image was set to 1°. The crystal-to-detector distance was 166 mm. The data were processed using iMOSFLM (Battye et al., 2011 ▶).

The crystals belonged to the monoclinic space group C2. A summary of the data statistics is presented in Table 1 ▶. Assuming two protein molecules in the asymmetric unit, the crystal volume per enzyme mass (V _M) and the solvent content were calculated to be 2.55 ų Da⁻¹ and 51.7%, respectively, which are within the frequently observed ranges for protein crystals (Matthews, 1968 ▶).

Table 1. Data-collection and processing statistics for Tx chicken fatty liver CR.

Values in parentheses are for the highest resolution shell.

Source	NE3A, Photon Factory
Wavelength (Å)	1.00
Rotation range per frame (°)	1
Total rotation range (°)	270
Exposure per frame (s)	1
Crystal-to-detector distance (mm)	166
Temperature (K)	100
Space group	C2
Unit-cell parameters (Å, °)	a = 104.26, b = 81.32, c = 77.27, β = 119.43
Resolution range (Å)	31.93–1.86 (1.96–1.86)
No. of measured reflections	253752
No. of unique reflections	47131
Multiplicity	5.4 (5.4)
Completeness (%)	99.4 (100.0)
R merge †	0.054 (0.089)
〈I/σ(I)〉	9.6 (6.8)
Mosaicity (°)	0.96

^† R _merge = .

Based on the structures of CRs from the fruit fly D. melanogaster (PDB entry 1sny; 36% identity; Sgraja et al., 2004 ▶), human (PDB entry 1wma; 33% identity; Tanaka et al., 2005 ▶), mouse (PDB entry 1cyd; 30% identity; Tanaka et al., 1996 ▶) and pig (PDB entry 2zat; 27% identity; Tanaka et al., 2008 ▶), we attempted to use the molecular-replacement method for phase calculation, but were unable to obtain useful data. We are currently attempting to prepare selenomethionine-substituted Tx chicken fatty liver CR.

To date, the crystal structures of CRs from fruit fly, human, mouse and pig have been determined, but no CRs from poultry have been structurally characterized. In this study, the first diffraction-quality crystals of chicken CR were obtained. We expect that elucidation of the three-dimensional structure of this enzyme will further increase our understanding of the structure–function relationship in Tx chicken fatty liver CR.