Binding of ß-D-glucosides and ß-D-mannosides by rice and barley ß-D-glycosidases with distinct substrate specificities.

Predominantly, rice Os3BGlu7 operates as a ß-d-glucosidase (EC 3.2.1.21), while barley HvBII acts as a ß-d-mannosidase (EC 3.2.1.25). Saturation transfer difference nuclear magnetic resonance (STD NMR) and transferred nuclear Overhauser effect (trNOE) spectroscopy in conjunction with quantum mechanics/molecular mechanics (QM/MM) modeling and docking at the 6-31+G* level were used to investigate binding of S- and O-linked gluco- and manno-configured aryl-ß-d-glycosides to Os3BGlu7 and HvBII. Kinetic analyses with 4-nitrophenyl ß-d-thioglucoside (4NP-S-Glc) and 4-nitrophenyl ß-d-thiomannoside (4NP-S-Man) indicated that the inhibitions were competitive with apparent K(i) constants of 664 and 710 µM for Os3BGlu7 and 95 and 266 µM for HvBII, respectively. The STD NMR and trNOESY experiments revealed that 4NP-S-Glc and 4NP-S-Man bound weakly in (4)C(1) conformations to Os3BGlu7; 4NP-S-Glc adopted (3)S(5) (B(3,O)) or (1)S(3) ((1,4)B) conformations, and 4NP-S-Man preferred (4)C(1) geometry, when bound to HvBII. The QM modeling and docking, based on GLIDE scores, predicted that 4NP-O-Glc, 4NP-O-Man, and 4NP-S-Man bound preferentially in (1)S(3) geometries to both enzymes, contrary to 4NP-S-Glc that could also adopt a (4)C(1) conformation, although in a "flipped-down" ring position. The experimental and computational data suggested that in glycoside recognition and substrate specificity of Os3BGlu7 and HvBII, a combination of the following determinants is likely to play key roles: (i) the inherent conformational and spatial flexibilities of gluco- and manno-configured substrates in the enzymes' active sites, (ii) the subtle differences in the spatial disposition of active site residues and their capacities to form interactions with specific groups of substrates, and (iii) the small variations in the charge distributions and shapes of the catalytic sites.

Rice family GH1 glycoside hydrolases with beta-D-glucosidase and beta-D-mannosidase activities.

Plant beta-D-mannosidases and a rice beta-D-glucosidase, Os3BGlu7, with weak beta-D-mannosidase activity, cluster together in phylogenetic analysis. To investigate the relationship between substrate specificity and amino acid sequence similarity in family GH1 glycoside hydrolases, Os3BGlu8 and Os7BGlu26, putative rice beta-D-glucosidases from this cluster, and a beta-D-mannosidase from barley (rHvBII), were expressed in Escherichia coli and characterized. Os3BGlu8, the amino acid sequence and molecular model of which are most similar to Os3BGlu7, hydrolysed 4-nitrophenyl-beta-D-glucopyranoside (4NPGlc) faster than 4-nitrophenyl-beta-D-mannopyranoside (4NPMan), while Os7BGlu26, which is most similar to rHvBII by these criteria, hydrolysed 4NPMan faster than 4NPGlc. All the enzymes hydrolyzed cellooligosaccharides with increased hydrolytic rates as the degree of polymerization increased from 3-6, but only rHvBII hydrolyzed cellobiose with a higher k(cat)/K(m) value than cellotriose. This was primarily due to strong binding of glucosyl residues at the+2 subsite for the rice enzymes, and unfavorable interactions at this subsite with rHvBII.