title: Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation.
abstract: The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1-->6)-D-Manp, alpha-D-Xylp-(1-->6)-D-Fruf, and alpha-d-Xylp-(1-->3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-d-Xylp-(1-->6)-D-Manp and alpha-d-Xylp-(1-->6)-D-Fruf inhibited intestinal alpha-glucosidases.
authors.affiliation: Division of Applied Bioscience, Hokkaido University, Sapporo, Japan.
authors.forename: Min-Sun
authors.initials: MS
authors.lastname: Kang
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.forename: Katsuro
authors.initials: K
authors.lastname: Yaoi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Mitsuishi
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Kim
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Doman
authors.initials: D
authors.lastname: Kim
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002475:Cellobiose / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006860:Hydrogen Bonding; D020128:Inhibitory Concentration 50; D007413:Intestinal Mucosa / Q000201:enzymology; D008956:Models, Chemical; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D009844:Oligosaccharides; C503200:YicI protein, E coli; D002475:Cellobiose; D014995:Xylosidases; D000520:alpha-Glucosidases