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Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (Glycine max Merr.) seedlings

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Abstract

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of β-(1→4)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [14C]Gal from UDP-[14C]Gal into intact and partially hydrolyzed lupin β-(1→4)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn2+ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M r>150,000) from lupin we used was very low but increased when the M r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M r (average M r 60,000) β-(1→4)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min−1 (mg protein)−1] than low-M r (average M r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M r galactan with endo-β-(1→4)-galactanase released mainly radioactive β-(1→4)-galactobiose and Gal, indicating that the transfer of [14C]Gal occurred through β-(1→4)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) β-(1→4)-galactooligomers with degree of polymerization at least 5. Gal7-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal8–10-PA.

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Abbreviations

AGP :

Arabinogalactan-protein

Ara :

Arabinose

DP :

Degree of polymerization

GalA :

Galacturonic acid

Gal n -PA :

Pyridylaminated β-(1→4)-galactooligosaccharides

GalT :

Galactosyltransferase

MALDI–TOF–MS :

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Rha :

Rhamnose

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Acknowledgements

We thank Dr. K. Hori of the Akita Research Institute of Food & Brewing, Akita, Japan, for carrying out the GC/MS spectrometry. We also thank Dr. H. Nakano, Osaka Municipal Research Institute, for the gift of endo-β-(1→4)-galactanase. This study was supported in part by a grant-in-aid (Glyco-technology Project) from the Ministry of Agriculture, Forestry, and Fisheries, Japan.

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  1. Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Saitama 338-8570, Japan

    Tomoyuki Konishi, Terukazu Mitome, Hiroyuki Hatsushika, Md. Ashraful Haque, Toshihisa Kotake & Yoichi Tsumuraya

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  1. Tomoyuki Konishi
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  2. Terukazu Mitome
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  3. Hiroyuki Hatsushika
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  4. Md. Ashraful Haque
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  5. Toshihisa Kotake
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  6. Yoichi Tsumuraya
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Correspondence to Yoichi Tsumuraya.

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Sugars described in this paper belong to the d-series unless otherwise noted

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Konishi, T., Mitome, T., Hatsushika, H. et al. Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (Glycine max Merr.) seedlings. Planta 218, 833–842 (2004). https://doi.org/10.1007/s00425-003-1163-8

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