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Binding of β-d-Glucosides and β-d-Mannosides by Rice and Barley β-d-Glycosidases with Distinct Substrate Specificities

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journal contribution
posted on 12.10.2010 by Teerachai Kuntothom, Michal Raab, Igor Tvaroška, Sebastien Fort, Salila Pengthaisong, Javier Cañada, Luis Calle, Jesús Jiménez-Barbero, James R. Ketudat Cairns, Maria Hrmova
Predominantly, rice Os3BGlu7 operates as a β-d-glucosidase (EC, while barley HvBII acts as a β-d-mannosidase (EC 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-β-d-glycosides to Os3BGlu7 and HvBII. Kinetic analyses with 4-nitrophenyl β-d-thioglucoside (4NP-S-Glc) and 4-nitrophenyl β-d-thiomannoside (4NP-S-Man) indicated that the inhibitions were competitive with apparent Ki 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 4C1 conformations to Os3BGlu7; 4NP-S-Glc adopted 3S5 (B3,O) or 1S3 (1,4B) conformations, and 4NP-S-Man preferred 4C1 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 1S3 geometries to both enzymes, contrary to 4NP-S-Glc that could also adopt a 4C1 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.