An isoflavone conjugate-hydrolyzing β-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, Toru Nakayama

Research output: Contribution to journalArticlepeer-review

80 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)30251-30259
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number40
DOIs
Publication statusPublished - 2006 Oct 6

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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