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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Molecular and Structural Characterization of Hexameric -D-Glucosidases in Wheat and Rye^[W]

Department of Applied Biology and Chemistry (M.S., K.Y., T.M.) and Department of Bioscience (S.Y.), Tokyo University of Agriculture, Setagaya, Tokyo 156–8502, Japan; Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606–8502, Japan (T.N.); and Department of Biotechnology, School of Biology Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649–6493, Japan (T.M., H.I.)

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-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. 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, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O--D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077693.

^* Corresponding author; e-mail sue{at}nodai.ac.jp; fax 81–3–5477–2619.

Received January 24, 2006; returned for revision April 21, 2006; accepted May 18, 2006.

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