title: Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition.
abstract: The rice BGlu1 β-D-glucosidase nucleophile mutant E386G is a glycosynthase that catalyzes the synthesis of cellooligosaccharides from α-d-glucopyranosyl fluoride (GlcF) donor and p-nitrophenyl (pNP) cellobioside (Glc2-pNP) or cello-oligosaccharide acceptors. When activity with other donors and acceptors was tested, the initial enzyme preparation cleaved pNP-β-D-glucopyranoside (Glc-pNP) and pNP-β-D-fucopyranoside (Fuc-pNP) to pNP and glucose and fucose, suggesting contamination with wild type BGlu1 β-glucosidase. The products from reaction of GlcF and Fuc-pNP included Fuc-β-(1→3)-Fuc-pNP, Glc-β-(1→3)-Fuc-pNP, and Fuc-β-(1→4)-Glc-β-(1→3)-Fuc-pNP, suggesting the presence of both wild type BGlu1 and its glycosynthase. Inhibition of the BGlu1 β-glucosidase activity within this preparation by cyclophellitol confirmed that the E386G glycosynthase preparation was contaminated with wild type BGlu1. Rice BGlu1 E386G-2, generated from a new construct designed to minimize back-mutation, showed glycosynthase activity without wild type hydrolytic or transglycosylation activity. E386G-2 catalyzed transfer of glycosyl residues from GlcF, α-L-arabinosyl fluoride, α-D-fucosyl fluoride, α-D-galactosyl fluoride, α-D-mannosyl fluoride, and α-D-xylosyl fluoride donors to Glc2-pNP acceptor. The synthetic products from the reactions of α-fucosyl fluoride and α-mannosyl fluoride donors were confirmed to result from addition of a β-(1→4)-linked glycosyl residue. Moreover, the E386G glycosynthase transferred glucose from GlcF donor to glucose, cellobiose, Glc-pNP, Fuc-pNP, pNP-β-D-galactopyranoside, and pNP-β-D-xylopyranoside acceptors, but little to pNP-β-D-mannopyranoside. Production of longer oligosaccharides occurred most readily on acceptors with an equatorial 4-OH. Elimination of wild type contamination thereby allowed a clear assessment of BGlu1 E386G glycosynthase catalytic abilities.
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, 111 University Avenue, Muang District, Nakhon Ratchasima 30000, Thailand.
authors.forename: Salila
authors.initials: S
authors.lastname: Pengthaisong
authors.forename: Chi-Fan
authors.initials: CF
authors.lastname: Chen
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: Buabarn
authors.initials: B
authors.lastname: Kuaprasert
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D002240:Carbohydrate Sequence; D003511:Cyclohexanols / Q000494:pharmacology*; D004187:Disaccharides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006031:Glycosylation; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000201:enzymology*
chemical_list: D003511:Cyclohexanols; D004187:Disaccharides; D004791:Enzyme Inhibitors; C063285:cyclophellitol; D008025:Ligases