Alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction.
Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively. PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not. No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside. This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.
Okuyama
Masayuki
M
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Mori
Haruhide
H
Watanabe
Kotomi
K
Kimura
Atsuo
A
Chiba
Seiya
S
D019943:Amino Acid Substitution; D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005964:Glucosyltransferases / Q000378:metabolism*; D006027:Glycosides / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
D000926:Anticodon; D017931:DNA Primers; D006027:Glycosides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; D000520:alpha-Glucosidases