pubs.acs.org/Biochemistry Published on Web 09/08/2010 r 2010 American Chemical Society Biochemistry 2010, 49, 8779–8793 8779 DOI: 10.1021/bi101112c Binding of β-D-Glucosides and β-D-Mannosides by Rice and Barley β-D-Glycosidases with Distinct Substrate Specificities † Teerachai Kuntothom, ‡,#,r Michal Raab, #,§ Igor Tvaro  ska, § Sebastien Fort, ) Salila Pengthaisong, ‡ Javier Ca~ nada, ^ Luis Calle, ^ Jes us Jim enez-Barbero, ^ James R. Ketudat Cairns, ‡ and Maria Hrmova* ,@,b ‡ School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand, § Department of Structure and Function of Saccharides, Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Bratislava, Slovak Republic, ) Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France, ^ Centro de Investigaciones Biol  ogicas, CSIC, Madrid, Spain, and @ Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, Australia. # These authors made equal contributions to this work. r Current address: Department of Chemistry, Mahasarakham University, Mahasarakham, Thailand. b Also affiliated with Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic. Received July 12, 2010; Revised Manuscript Received September 7, 2010 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 6-31þG* 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-thioman- noside (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. The glycoside hydrolase GH1 family includes enzymes with approximately 20 known substrate specificities (1), including β-D-glucosidases (EC 3.2.1.21), 6-phospho-β-D-glucosidases (EC 3.2.1.86), β-D-mannosidases (EC 3.2.1.25), β-D-galactosidases (EC 3.2.1.23), β-D-glucuronidases (EC 3.2.1.31), and others (2). A detailed examination of the substrate specificity of the barley β-D-glucosidase isoenzyme βII (HvBII) (3, 4), also designated HvβMannos1 (5), revealed that it exhibits a marked preference for manno-oligosaccharides and that the rate of hydrolysis increases with the degree of polymerization of both cello- and manno-oligosaccharides (3-6). Hence, the substrate specificity and action patterns of HvBII are characteristic of an oligosaccharide exohydrolase, rather than of an enzyme with a preference for low- molecular mass cello-oligosaccharides. Similar conclusions were drawn for an Os3BGlu7 β-D-glucosidase from rice (also called BGlu1), although binding energies at individual subsites differ somewhat (6, 7). Both plant enzymes are capable of catalyzing transglycosylation reactions with 4NP-O-Glc 1 (3, 4, 6, 7), but not with 4NP-O-Man. The presence of an extended series of subsites in these two plant β-D-glycosidases indicates that their biological † This work was supported by grants from the Australian Research Council to M.H. and from the Thailand Research Fund (BRG5080007) to J.R.K.C. T.K. was sponsored by The Institute for the Promotion of Teaching Science and Technology of Thailand. M.R. and I.T. are thankful for support from the Science and Technology Assistance Agency under Contract APVV-0607-07. J.C., L.C., and J.J.-B. thank the Ministerio de Ciencia e Innovacion for support (CTQ2009-08536). *To whom correspondence should be addressed. Telephone: þ61 8 8303 7160. Fax: þ61 8 8303 7102. E-mail: maria.hrmova@adelaide.edu.au. 1 Abbreviations: CAZy, Carbohydrate-Active enZymes; DP, degree of polymerization; CORCEMA, Complete Relaxation and Conforma- tional Exchange Matrix; DPFGSE, double pulse field-gradient spin- echo; EA, catalytic nucleophile; EB, catalytic acid/base; ESP, electro- static potential; GH, glycoside hydrolase; HDO, hydrogen/deuterium water; ISPA, isolated spin-pair approximation; NOEs, nuclear Over- hauser effects; PDB, Protein Data Bank; rmsd, root-mean-square deviation; QM/MM, quantum mechanics/molecular mechanics; STD NMR, saturation transfer difference nuclear magnetic resonance; trNOESY, transferred nuclear Overhauser effect spectroscopy; 1D, one-dimensional; 2D, two-dimensional; 3D, three-dimensional; 4NP, 4-nitrophenyl; 4NP-O-Glc, 4-nitrophenyl β-D-glucopyranoside; 4NP- O-Man, 4-nitrophenyl β-D-mannopyranoside; 4NP-S-Glc, 4-nitrophe- nyl β-D-thioglucopyranoside; 4NP-S-Man, 4-nitrophenyl β-D-thioman- nopyranoside.