The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing β-glucosidase with significant thioglucohydrolase activity.

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) β-glucosidase, hydrolyzes β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the β-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a (4)C(1) chair conformation, while that of the noncovalently bound DNP2FG had a (1)S(3) skew boat, consistent with hydrolysis via a (4)H(3) half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 β-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.