Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases.
Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) 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.
Daiyasu
Hiromi
H
The Center for Advanced Medical Engineering and Informatics, Osaka University, Toyonaka, Osaka, Japan. daiyasu@ist.osaka-u.ac.jp
Saino
Hiromichi
H
Tomoto
Hiroo
H
Mizutani
Masaharu
M
Sakata
Kanzo
K
Toh
Hiroyuki
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004186:Disaccharidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D027925:Viburnum / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D018744:DNA, Plant; D004187:Disaccharides; D004186:Disaccharidases; D006026:Glycoside Hydrolases