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Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition

TitleRice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition
Publication TypeJournal Article
Year of Publication2012
AuthorsPengthaisong, S, Chen, C-F, Withers, SG, Kuaprasert, B, Cairns, JRKetudat
Date PublishedMAY 1

The rice BGlu1 beta-D-glucosidase nucleophile mutant E386G is a glycosynthase that catalyzes the synthesis of cellooligosaccharides from alpha-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-beta-D-glucopyranoside (Glc-pNP) and pNP-beta-D-fucopyranoside (Fuc-pNP) to pNP and glucose and fucose, suggesting contamination with wild type BGlu1 beta-glucosidase. The products from reaction of GlcF and Fuc-pNP included Fuc-beta-(1 -> 3)-Fuc-pNP, Glc-beta-(1 -> 3)Fuc-pNP, and Fuc-beta-(1 -> 4)-Glc-beta-(1 -> 3)-Fuc-pNP, suggesting the presence of both wild type BGlu1 and its glycosynthase. Inhibition of the BGlu1 beta-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, alpha-L-arabinosyl fluoride, alpha-D-fucosyl fluoride, alpha-D-galactosyl fluoride, alpha-D-mannosyl fluoride, and alpha-D-xylosyl fluoride donors to Glc2-pNP acceptor. The synthetic products from the reactions of alpha-fucosyl fluoride and alpha-mannosyl fluoride donors were confirmed to result from addition of a beta-(1 -> 4)-linked glycosyl residue. Moreover, the E386G glycosynthase transferred glucose from GlcF donor to glucose, cellobiose, Glc-pNP, Fuc-pNP, pNP-beta-D-galactopyranoside, and pNP-beta-D-xylopyranoside acceptors, but little to pNP-beta-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. (C) 2012 Elsevier Ltd. All rights reserved.