Binding of β-D-glucosides and β-D-mannosides by rice and barley β-D-glycosidases with distinct substrate specificities
Date
2010
Authors
Kuntothom, T.
Raab, M.
Tvaroska, I.
Fort, S.
Pengthaisong, S.
Canada, J.
Calle, L.
Jiminez-Barbero, J.
Cairns, J.
Hrmova, M.
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Biochemistry, 2010; 49(40):8779-8793
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Teerachai Kuntothom, Michal Raab, Igor Tvaroska, Sebastien Fort, Salila Pengthaisong, Javier Cañada, Luis Calle, Jesús Jiménez-Barbero, James R. Ketudat Cairns, and Maria Hrmova
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Abstract
Predominantly, rice Os3BGlu7 operates as a β-d-glucosidase (EC 3.2.1.21), while barley HvBII acts as a β-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-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 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.
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Copyright © 2010 American Chemical Society