title: The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase.
abstract: Rice BGlu1 β-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without α-glucosyl fluoride were solved and the α-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β-glucosidase hydrolyzed cellotetraose and cellopentaose with k(cat) /K(m)  values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP-cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.
authors.affiliation: Schools of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Salila
authors.initials: S
authors.lastname: Pengthaisong
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: Buabarn
authors.initials: B
authors.lastname: Kuaprasert
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.forename: James R Ketudat
authors.initials: JR
authors.lastname: Cairns
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D009844:Oligosaccharides / Q000096:biosynthesis / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D013780:Tetroses / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D013780:Tetroses; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D001617:beta-Glucosidase