Structural and enzymatic characterization of Os3BGlu6, a rice beta;-glucosidase hydrolyzing hydrophobic glycosides and (1→3)- and (1→2)-linked disaccharides

Abstract

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 ρ-nitrophenyl (ρNP)-β-D-fucoside (kcat/Km = 67 mM-1 s-1), ρNP-β-D-glucoside (kcat/Km = 6.2 mM-1 s-1), and ρNP-β-D-galactoside (kcat/Km = 1.6 mM-1s-1) efficiently but had little activity toward other pNP glycosides. It also had high activity toward η-octyl- β-D-glucoside and β-(1→3)- and β-(1→2)-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, η-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-(1→4)-linked oligosaccharides and interact with the hydrophobic aglycone of η-octyl-β-D-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides. © 2009 American Society of Plant Biologists.