Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

6A/2005G269

Purification, crystallization and preliminary X-ray analysis of a hexameric βglucosidase from wheat
1

Masayuki SUE1, Jun-ichi KOUYAMA2, Yasuyuki SASAKI2, Shunsuke YAJIMA*2
Department of Applied Biology and Chemistry, and 2Department of Bioscience, Tokyo University ofAgriculture, Setagaya-ku, Tokyo 156-8502, Japan

Introduction
β-Glucosidase (EC. 3.2.1.21) is one of the largest
groups in the glycoside hydrolase families, and is amember of families 1 or 3 [1]. These enzymes areresponsible for cleavage of the β-glucosidic linkage
between two (or more) sugars, or between a sugar and an
aglycone. In plants, β-glucosidases are involved inlignification, regulation of the physiological activity ofcytokinin, regulation of the biosynthesis of IAA, andchemical defense against pathogens and herbivores. Many
secondary products in plants exist in a form of glucoconjugate with one or two glucose units attaching to theirhydroxy or thiol groups. Since glucosylation changes
physiological activity, chemical stability, and solubility towater, β-glucosidases play important roles by cleaving
glucosidic bonds to release monosaccharides and active
aglycones such as cyanogenic compounds, flavonoids,and hydroxamic acids. The glucosidases involved in thehydrolysis of plant secondary metabolites are members offamily 1 glycohydrolase.
Recently, we cloned three β-glucosidase genes from
wheat seedlings and expressed them in E. coli. The
recombinant glucosidases formed a hexamer, the activeform, consisting of 64 kDa. The hexamer was easily
dissociated into monomer, resulting in loss of activity.
The crystal structures of family 1 β-glucosidases havebeen isolated from four plant species: white clover, whitemustard, maize, and sorghum. The wheat enzyme,
however, differs in its active oligomeric structure, since
wheat glucosidase must be hexameric to be active as ahydrolase whereas the other plant glucosidases function
as dimers. The crystal structures of family 1 βglucosidase from some bacteria have been solved as
octamers. The wheat enzyme, however, shares lower
homologies (~35%) with bacterial ones than those withplants (~60%). Thus, it is of interest to compare themonomer association properties from quaternary
structures. We therefore aimed to crystallize wheat βglucosidase as a hexamer in order to clarify the relation
between its structure and the activity.
Materials and Methods
The initial crystallization conditions were screened by
Hampton Research Crystal Screens I and II; the final
condition was 10 mM HEPES (pH 7.2), 1 M LiSO4, and150 mM NaCl as the reservoir buffer. All crystallization
experiments were performed using the hanging-drop
vapor diffusion method in a 24-well tissue-culture VDX
plate at 293 K. Drops consisting of 1 µl protein (3.7mg/ml) and 1 µl reservoir solution were used for theinitial screening, and 1.5 µl and 1.5 µl were used for thefinal condition.
To obtain the complex of TaGlu1b and its substrate
aglycone, DIMBOA, the crystals were soaked in thecrystallization buffer with 0.5 mM DIMBOA and 30%
glycerol as a cryoprotectant for 15 min, and were thencooled in a nitrogen stream at 100 K. The diffraction data
were processed using the HKL2000 program.
Results and Discussion
From the initial screening, crystals were obtained fromboth 0.1 M HEPES pH 7.5, 2 M ammonium sulfate, 2%
PEG400, and 10 mM HEPES pH 7.2, 1 M LiSO4, 150
mM NaCl. The first set of conditions produced only tinycrystals, so we proceeded to the latter one. The crystals
were typically 0.2 mm in their longest dimension, with amaximum of around 0.3 mm; we used the largest crystals
for data analysis. TaGlu1b crystals complexed withDIMBOA were obtained by soaking, and diffracted to 1.7
Å on beamline BL-6A at Photon Factory. However,crystals without the substrate diffracted less than 2.0 Å.
The mosaicity of the crystals was very low (~0.12), andRmerge was 0.079 with high redundancy (20).
A molecular replacement solution, using the βglucosidase from sorghum (PDB code 1v08) as the searchmodel, has been obtained using the program Molrep.
From this solution, the asymmetric unit contains one
monomer, and the solvent content was 72.1%, which isrelatively high in spite of the higher resolution [2]. Modelbuilding and refinement of this structure are under way.
Several structures of O-β-glucosidase from plants,
including white clover, maize, and sorghum, have beeninferred. The resolution obtained in the present study isbetter, however, and promises to give better
understanding of the relation between structure andactivity of O-β-glucosidase.
References[1] B. Henrissat, Biochem. J. 280, 309 (1991).
[2] M. Sue et al., Acta Crystallogr. F61, 864 (2005).
* yshun@nodai.ac.jp

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