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Journal of Biochemistry Advance Access published online on July 30, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn095
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© 2008 The Japanese Biochemical Society

Computational and Experimental Analyses of Furcatin Hydrolase for Substrate Specificity Studies of Disaccharide-specific Glycosidases

Hiromi Daiyasu1,4, Hiromichi Saino2, Hiroo Tomoto2, Masaharu Mizutani2, Kanzo Sakata2 and Hiroyuki Toh3

1. The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
2. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
3. Division of Bioinformatics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

4. Corresponding author: Dr. Hiromi Daiyasu, daiyasu{at}ist.osaka-u.ac.jp, Tel: +81-6-6850-6603, Fax: +81-6-6850-6602

Received May 31, 2008; Accepted July 5, 2008


  Abstract

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific β-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyze the β-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyze monosaccharide glycosides. We analyzed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.16 [EC] 1) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.

Key Words: β-glucosidase, disaccharide glycoside, furcatin hydrolase, homology modeling, mutagenesis


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