Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation |
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Authors: | Chuenchor Watchalee Pengthaisong Salila Robinson Robert C Yuvaniyama Jirundon Oonanant Worrapoj Bevan David R Esen Asim Chen Chun-Jung Opassiri Rodjana Svasti Jisnuson Cairns James R Ketudat |
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Affiliation: | 1 Schools of Biochemistry & Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 2 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673 Singapore 3 Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 4 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA 5 Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA 6 Life Science Group, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan R.O.C. |
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Abstract: | The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates. |
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Keywords: | ABTS, 2,2&prime -azinobis-3-ethylbenzthiazolinesulfonic acid CC, correlation coefficient DLS, dynamic light-scattering DIMBOAGlc, 2-O-β- smallcaps" >d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one DNP2FG, 2-deoxy-2-fluoro-β- smallcaps" >d-glucoside DP, degree of polymerization free BGlu1, rice BGlu1 beta-glucosidase with no substrate-based inhibitor G2F, 2-deoxy-2-fluoro-β- smallcaps" >d-glucoside GH1, glycosyl hydrolase family 1 IMAC, immobilized metal affinity chromatography LGA, Lamarckian genetic algorithm Mes, 2-morpholinoethanesulfonic acid NCS, non-crystallographic symmetry PDB, Protein Data Bank PEG MME, polyethylene glycol monomethyl ether pNP, p-nitrophenol pNPG, p-nitrophenyl β- smallcaps" >d-glucopyranoside PpBglB, Paenibacillus polymyxa β-glucosidase BglB RMSD, root-mean-square deviation SbDhr1, Sorghum bicolor β-glucosidase isozyme 1 V0, initial velocity ZmGlu1, Zea mays β-glucosidase isozyme 1 |
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