Alpha-glucosidase mutant catalyzes 'alpha-glycosynthase'-type reaction

• Published in: Bioscience, biotechnology, and biochemistry Vol. 66; no. 4; pp. 928 - 933
• Main Authors: Okuyama, M. (Hokkaido Univ., Sapporo (Japan)), Mori, H, Watanabe, K, Kimura, A, Chiba, S
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 2002; Japan Society for Bioscience Biotechnology and Agrochemistry
• Subjects: alpha-Glucosidases - genetics; alpha-Glucosidases - metabolism; Amino Acid Substitution; Anticodon - genetics; Base Sequence; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Catalysis; CATALYTIC ACTIVITY; catalytic nucleophile mutant; DNA Primers; Fundamental and applied biological sciences. Psychology; GLUCOSIDASES; Glucosyltransferases - metabolism; GLYCINE (AMINO ACID); Glycosides - chemical synthesis; glycosynthase; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; LIGASES; Methods. Procedures. Technologies; Mutagenesis, Site-Directed; MUTANTS; oligosaccharide production; OLIGOSACCHARIDES; Polymerase Chain Reaction; Protein engineering; Recombinant Proteins - metabolism; SCHIZOSACCHAROMYCES; Schizosaccharomyces - enzymology; α-glucosidase; Enzymatic synthesis; Enzyme; Oligosaccharide; Site directed mutagenesis; Fungi; α-Glucosidase; Glycosidases; Schizosaccharomyces pombe; Hydrolases; Ascomycetes; Mutation; O-Glycosidases; Thallophyta; Enzymatic reaction
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.66.928

Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces potnbe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an a-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.