Y. Ahn, M. Mizutani, H. Saino
May 28, 2004
Citations
1
Influential Citations
24
Citations
Journal
Journal of Biological Chemistry
Abstract
Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-β-d-apiofuranosyl-β-d-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf β-primeverosidase, which is another disaccharide glycosidase specific to β-primeverosides (6-O-β-d-xylopyranosyl-β-d-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant β-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the β-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed β-primeverosides and β-vicianoside (6-O-α-l-arabinopyranosyl-β-d-glucopyranoside) but poorly hydrolyzed β-gentiobiosides (6-O-β-d-glucopyranosyl-β-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl β-d-glucopyranoside containing the same aglycone as furcatin but little activity toward the other β-d-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant β-glucosidases revealed that FH clusters with β-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.