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J. Biol. Chem., Vol. 281, Issue 40, 30251-30259, October 6, 2006

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An Isoflavone Conjugate-hydrolyzing -Glucosidase from the Roots of Soybean (Glycine max) Seedlings

PURIFICATION, GENE CLONING, PHYLOGENETICS, AND CELLULAR LOCALIZATION^*

Hirokazu Suzuki, Seiji Takahashi, Ryoko Watanabe, Yusuke Fukushima, Naoki Fujita, Akio Noguchi, Ryusuke Yokoyama, Kazuhiko Nishitani, Tokuzo Nishino, and Toru Nakayama¹

From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai 980-8579 and the Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba-yama 6-3, Sendai 980-8578, Japan

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe 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 -glucosidase. Here we report on the purification and cDNA cloning of this important -glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing -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--D-glucoside) to produce free genistein (k_cat, 98 s^-1; K_m, 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 -glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

Received for publication, June 15, 2006 , and in revised form, August 2, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB259819 [GenBank] .

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," Table 1S, Fig. 1S and Fig. 2S, and supplemental Refs. 1-9.

¹ To whom correspondence should be addressed. Fax: 81-22-795-7270; E-mail: nakayama{at}seika.che.tohoku.ac.jp.

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