Endo-β-1,3-galactanase from Winter Mushroom Flammulina velutipes

Masahiro Samejima,Hitomi Ichinose,Kiyohiko Igarashi,Yuta Degi,Ryohei Takata,Kiminari Kitazawa,Maki Ishiguro,Yoichi Tsumuraya,Toshihisa Kotake,Satoshi Kaneko,Naohiro Hirata

doi:10.1074/jbc.m111.251736

Masahiro Samejima, Hitomi Ichinose + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m111.251736

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Abstract

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.

Highlights

Active ingredient in the herbal medicine Juzen-Taiho-To [4, 5]
Arabinogalactan proteins (AGPs) are differentiated by their core proteins, the AG moieties have a common overall structure, that is, ␤-1,3galactan main chains to which ␤-1,6-galactan side chains are attached through O-6
The ABEE-derivatized sugars were analyzed on a highperformance liquid chromatography (HPLC) system equipped with a TSKgel Amide-80 (4.6 ϫ 250 mm; Tosoh, Tokyo, Japan) column as described previously [18]

Summary

EXPERIMENTAL PROCEDURES

Culture of Native F. velutipes—F. velutipes (NBRC 7663) was distributed from NITE Biological Resource Center (Kisarazu, Japan). Purification of the Native Enzyme—Native FvEn3GAL was purified from the culture medium of F. velutipes mycelia. Enzyme Assay—The activity of the native enzyme toward polysaccharides was measured using reaction mixtures (total volume, 0.1 ml) consisting of 0.1% (w/v) polysaccharide, 50 mM acetate buffer (pH 5.0), and the enzyme. The enzymatic activity toward Galn was measured using reaction mixtures (total volume, 8 ␮l) consisting of the enzyme, 2.5 mM oligosaccharides, and 10 mM acetate buffer (pH 5.0). Analysis of Mode of Action—The mode of hydrolysis action was analyzed with a reaction mixture (total volume, 6 ␮l) containing the native enzyme, 1 mM ␤-1,3-Gal, and 50 mM acetate buffer (pH 5.0). The ABEE-derivatized sugars were analyzed on a highperformance liquid chromatography (HPLC) system equipped with a TSKgel Amide-80 (4.6 ϫ 250 mm; Tosoh, Tokyo, Japan) column as described previously [18]

RESULTS

Xyloglucan from tamarind

DISCUSSION