Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 {beta}-glucosidase glycosynthase mutants.
Rice BGlu1 beta-glucosidase is a glycosyl hydrolase family 1 enzyme that acts as an exoglucanase on beta-(1,4)- and short beta-(1,3)-linked gluco-oligosaccharides. Mutations of BGlu1 beta-glucosidase at glutamate residue 414 of its natural precursor destroyed the enzyme's catalytic activity, but the enzyme could be rescued in the presence of the anionic nucleophiles such as formate and azide, which verifies that this residue is the catalytic nucleophile. The catalytic activities of three candidate mutants, E414G, E414S, and E414A, in the presence of the nucleophiles were compared. The E414G mutant had approximately 25- and 1400-fold higher catalytic efficiency than E414A and E414S, respectively. All three mutants could catalyze the synthesis of mixed length oligosaccharides by transglucosylation, when alpha-glucosyl fluoride was used as donor and pNP-cellobioside as acceptor. The E414G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than that of E414S and E414A, respectively, and gave yields of up to 70-80% insoluble products with a donor-acceptor ratio of 5:1. (13)C-NMR, methylation analysis, and electrospray ionization-mass spectrometry showed that the insoluble products were beta-(1,4)-linked oligomers with a degree of polymerization of 5 to at least 11. The BGlu1 E414G glycosynthase was found to prefer longer chain length oligosaccharides that occupy at least three sugar residue-binding subsites as acceptors for productive transglucosylation. This is the first report of a beta-glucansynthase derived from an exoglycosidase that can produce long-chain cello-oligosaccharides, which likely reflects the extended oligosaccharide-binding site of rice BGlu1 beta-glucosidase.
Hommalai
Greanggrai
G
Center for Protein Structure and Function, Mahidol University, Bangkok 10400, Thailand.
Withers
Stephen G
SG
Chuenchor
Watchalee
W
Cairns
James R Ketudat
JR
Svasti
Jisnuson
J
D002241:Carbohydrates / Q000737:chemistry; D002621:Chemistry / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005964:Glucosyltransferases / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008956:Models, Chemical; D009844:Oligosaccharides / Q000737:chemistry*; D012275:Oryza / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D001617:beta-Glucosidase / Q000235:genetics* / Q000502:physiology
D002241:Carbohydrates; D009844:Oligosaccharides; D005964:Glucosyltransferases; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase