Substitution of the catalytic acid-base Glu237 by Gln suppresses hydrolysis during glucosylation of phenolic acceptors catalyzed by Leuconostoc mesenteroides sucrose phosphorylase

Stereoselective glycosylation of a phenolic hydroxyl is a key transformation in the (bio)synthesis of natural products. Biocatalytic transglycosylation usually provides the desired glycosidic product in exquisite anomeric purity. However, loss of substrate and product to hydrolysis often limits application of the method. Kinetic studies and in situ proton NMR analysis of reaction time courses were used here to characterize glucosylation of substituted phenol acceptors by Leuconostoc mesenteroides sucrose phosphorylase in the presence of alpha-D-glucose 1-phosphate (alpha G1P) as donor substrate. In the wild-type enzyme, hydrolysis of the sugar 1-phosphate strongly prevailed (about 10-fold, similar to 1.6 U/mg) over glucosyl transfer to the 2,6-difluorophenol acceptor (similar to 0.17 U/mg) used. A mutated phosphorylase in which the catalytic acid-base Glu(237) had been replaced by Gln (E237Q) did not display hydrolase activity under trans-glucosylation conditions and therefore provided substantial (similar to 7-fold) enhancement of transfer yield. Utilization of the donor substrate was however slowed down (about 400-fold, similar to 0.004 U/mg) in E237Q as compared to wild-type enzyme (similar to 1.6 U/mg). In a series of mono- and disubstituted phenols differing in hydroxyl pK(a) between 7.02 and 8.71, the transferase activity of E237Q was found to be dependent on steric rather than electronic properties of the acceptor used. Both wild-type and mutated enzyme employed 4-nitrophenyl-alpha-D-glucopyranoside (4-NPG) as a slow artificial substrate for phosphorolysis and hydrolysis (native: similar to 0.12 U/mg; E237Q: similar to 0.02 U/mg). (C) 2010 Elsevier B.V. All rights reserved.