TY - JOUR
T1 - An isoflavone conjugate-hydrolyzing β-glucosidase from the roots of soybean (Glycine max) seedlings
T2 - Purification, gene cloning, phylogenetics, and cellular localization
AU - Suzuki, Hirokazu
AU - Takahashi, Seiji
AU - Watanabe, Ryoko
AU - Fukushima, Yusuke
AU - Fujita, Naoki
AU - Noguchi, Akio
AU - Yokoyama, Ryusuke
AU - Nishitani, Kazuhiko
AU - Nishino, Tokuzo
AU - Nakayama, Toru
PY - 2006/10/6
Y1 - 2006/10/6
N2 - Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plant-microbe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing β-glucosidase. Here we report on the purification and cDNA cloning of this important β-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing β-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6″-O-malonyl-β-D-glucoside) to produce free genistein (kcat, 98 s-1; Km, 25 μM at 30°C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous β-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.
AB - Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plant-microbe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing β-glucosidase. Here we report on the purification and cDNA cloning of this important β-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing β-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6″-O-malonyl-β-D-glucoside) to produce free genistein (kcat, 98 s-1; Km, 25 μM at 30°C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous β-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.
UR - http://www.scopus.com/inward/record.url?scp=33749563523&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33749563523&partnerID=8YFLogxK
U2 - 10.1074/jbc.M605726200
DO - 10.1074/jbc.M605726200
M3 - Article
C2 - 16891302
AN - SCOPUS:33749563523
VL - 281
SP - 30251
EP - 30259
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 40
ER -