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J. Biol. Chem., Vol. 279, Issue 22, 23405-23414, May 28, 2004

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Furcatin Hydrolase from Viburnum furcatum Blume Is a Novel Disaccharide-specific Acuminosidase in Glycosyl Hydrolase Family 1^*

Young Ock Ahn, Masaharu Mizutani, Hiromichi Saino, and Kanzo Sakata

From the Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O--D-apiofuranosyl--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 -primeverosidase, which is another disaccharide glycosidase specific to -primeverosides (6-O--D-xylopyranosyl--D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant -glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a K_m value of 2.2 mM and specifically hydrolyzed the -glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed -primeverosides and -vicianoside (6-O--L-arabinopyranosyl--D-glucopyranoside) but poorly hydrolyzed -gentiobiosides (6-O--D-glucopyranosyl--D-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl -D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other -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 -glucosidases revealed that FH clusters with -primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.

Received for publication, October 16, 2003 , and in revised form, February 18, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB122081.

^* This work was supported in part by Grant-in-aid (B)(2)13460049 from the Ministry of Education, Science, Sports, and Culture of Japan (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-774-38-3232; Fax: 81-774-38-3229; E-mail: mizutani{at}scl.kyoto-u.ac.jp.

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