Photon Factory Activity Report 2014 #32 (2015) B

5A/ 2014G502, 2013G003

Crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl
reductase specifically expressed in the thyroidectomized chicken fatty liver
Kazunari Yoneda*1, Haruhiko Sakuraba2, Tomohiro Araki1, Toshihisa Ohshima3
1
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, Japan2
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393
Ikenobe, Miki-cho, Kita-gun, Kagawa761-0795, Japan3
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan1. Introduction
In 2003, Shibata et al. have demonstrated that thefunctionally unknown 29 kDa protein is specifically
expressed in thyroidectomized (Tx) chicken fatty liver.
On the basis of genome information of the chicken(Gallus gallus), we recently identified the gene encoding
the protein.
From comparison of the amino-acidsequence of this protein with those of the homologues
whose crystal structures have been determined to date, we
found that the protein exhibits the highest identity (36%)
with a CR from fruit-fly Drosophila melanogaster (PDBentry 1sny; Sgraja et al., 2004) and contains the twoconsensus sequences (S-YXXXK, GXXXGXG) of the SDRfamily. Moreover, we succeeded in the expression of thegene in E. coli and confirmed that the gene product surely
exhibits CR activity [1]. Since the enzyme shows rather
low sequence identities with 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, andmammalian CRs. The physiological function of the CRin Tx chicken fatty liver is currently unknown. The
enzyme probably contribute to metabolism ofaccumulated ketone body derived from fatty acid.
Structural analysis of the enzyme may provide important
information about its physiological substrate and function.
In this study, we describe crystallization and preliminary
X-ray analysis of NAD(P)H-dependent CR which isspecifically expressed in the fatty liver ofthyroidectomized chicken.
2. Experiment
Initial screening for crystallization was carried out withIndex (Hampton Research, USA) at 293 K using thesitting-drop vapor diffusion method.
The NAD(P)H-dependent CR crystal was flash-cooledin liquid nitrogen at 100 K. Diffraction data werecollected at 1.98 Å resolution using monochromatedradiation of wavelength 1.0 Å and an ADSC CCD
detector system on the 5A beamline at the Photon Factory,
Tsukuba, Japan. The oscillation angle per image was set
to 1˚. The crystal-to-detector distance was 249 mm. The
data were processed using HKL-2000.
3. Results and Discussion
The diffraction-quality crystal (maximum dimensionsof 0.6  0.3  0.05 mm; Fig. 1) was obtained within twoweeks using a reservoir solution composed of 23% PEG
5000MME, 100 mM Bis-Tris buffer (pH 6.5).
The crystals belonged to the trigonal space group
P3221 (Fig. 2). Assuming two protein molecules in theasymmetric unit, the crystal volume per enzyme mass(VM) and the solvent content were calculated to be 2.57
Å3 Da-1 and 51.7%, respectively, which are within thefrequently observed ranges for protein crystals.

Fig.1 Trigonal crystal of Tx chicken fatty liver CR.
Maximum dimensions of the crystal are 0.6  0.3  0.05mm.

Fig.2 X-ray diffraction of a Tx chicken fatty liver CR.
Reference[1] K. Yoneda et al., Acta Crystallographica Section F.
(2012) F68, 1568-1570.
* kyoneda@agri.u-tokai.ac.jp