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

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.