Received January 24, 2006
Returned for revision March 5, 2006
Accepted May 18, 2006
Molecular and Structural Characterization of Hexameric 
-D-Glucosidases in Wheat and Rye
Masayuki Sue *, Kana Yamazaki , Shunsuke Yajima , Taiji Nomura , Tetsuya Matsukawa , Hajime Iwamura , and Toru Miyamoto
Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan
Department of Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan
Department of Biotechnology, School of Biology Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, Japan
* Corresponding author; email: sue{at}nodai.ac.jp.
The wheat (Triticum aestivum) and rye (Secale cereale) 
-D-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding -D-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-hour-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct from TaGlu1a and TaGlu1c. Furthermore, co-expression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and hetero-hexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 Å. The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, Ser464 and Leu465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-Glc (2-O--D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-Glc (7-demethoxy-DIMBOA-Glc).
