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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (13)- and (12)-Linked Disaccharides^{1,[C],[W],[OA]}

Schools of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand (S.S., R.O., J.K.C.); National Agricultural Research Center for the Hokkaido Region, Sapporo 062–8555, Japan (T.A.); and Synchrotron Light Research Institute, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand (B.K.)

Glycoside hydrolase family 1 (GH1) β-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice β-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-β-D-fucoside (k_cat/K_m = 67 mM^–1 s^–1), pNP-β-D-glucoside (k_cat/K_m = 6.2 mM^–1 s^–1), and pNP-β-D-galactoside (k_cat/K_m = 1.6 mM^–1s^–1) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-β-D-glucoside and β-(13)- and β-(12)-linked disaccharides and was able to hydrolyze apigenin β-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-β-D-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 Å resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended β-(14)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-β-D-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: James Ketudat Cairns (cairns{at}sut.ac.th).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

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

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www.plantphysiol.org/cgi/doi/10.1104/pp.109.139436

^* Corresponding author; e-mail cairns{at}sut.ac.th.

Received April 3, 2009; accepted July 2, 2009; published July 8, 2009.