Structural and enzymatic characterization of Os3BGlu6, a rice beta-glucosidase hydrolyzing hydrophobic glycosides and (1->3)- and (1->2)-linked disaccharides.
Glycoside hydrolase family 1 (GH1) beta-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 beta-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-beta-d-fucoside (k(cat)/K(m) = 67 mm(-1) s(-1)), pNP-beta-d-glucoside (k(cat)/K(m) = 6.2 mm(-1) s(-1)), and pNP-beta-d-galactoside (k(cat)/K(m) = 1.6 mm(-1)s(-1)) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-beta-d-glucoside and beta-(1-->3)- and beta-(1-->2)-linked disaccharides and was able to hydrolyze apigenin beta-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-beta-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 A 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 beta-(1-->4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-beta-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.
Seshadri
Supriya
S
School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Akiyama
Takashi
T
Opassiri
Rodjana
R
Kuaprasert
Buabarn
B
Cairns
James Ketudat
JK
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D006027:Glycosides / Q000378:metabolism*; D008958:Models, Molecular; D012275:Oryza / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D004187:Disaccharides; D006027:Glycosides; D010940:Plant Proteins; D001617:beta-Glucosidase