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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Molecular Cloning of a -Galactosidase from Radish That Specifically Hydrolyzes -(13)- and -(16)-Galactosyl Residues of Arabinogalactan Protein¹

Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Sakura-ku, Saitama 338–8570, Japan (T.K., S.D., T.K., Y.T.); Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305–8642, Japan (S.K.); Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (K.I., M.S.); and Yakult Central Institute for Microbiological Research, Tokyo 186–8650, Japan (Y.W., K.K.)

A basic -galactosidase with high specificity toward -(13)- and -(16)-galactosyl residues was cloned from radish (Raphanus sativus) plants by reverse transcription-PCR. The gene, designated RsBGAL1, contained an open reading frame consisting of 2,532 bp (851 amino acids). It is expressed in hypocotyls and young leaves. RsBGAL1 was highly similar to -galactosidases having exo--(14)-galactanase activity found in higher plants and belongs to family 35 of the glycosyl hydrolases. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to homogeneity. The recombinant enzyme specifically hydrolyzed -(13)- and -(16)-galactooligosaccharides, the same substrates as the native enzyme isolated from radish seeds (Sekimata et al., 1989). It split off about 90% of the carbohydrate moieties of an arabinogalactan protein extracted from radish roots in concerted action with microbial -L-arabinofuranosidase and -glucuronidase. These results suggest that RsBGAL1 is a new kind of -galactosidase with different substrate specificity than other -galactosidases that exhibit exo--(14)-galactanase activity. The C-terminal region (9.6 kD) of RsBGAL1 is significantly similar to the Gal lectin-like domain, but this region is not retained in the native enzyme. Assuming posttranslational processing of RsBGAL1 with elimination of the Gal lectin-like domain results in a protein consisting of two subunits with molecular masses of 46 and 34 kD (calculated from the RsBGAL1 gene sequence). This is in good agreement with the SDS-PAGE and matrix-assisted laser desorption/ionization-time-of flight mass spectrometry measurements for subunits of the native enzyme (45 and 34 kD) and may thus partially explain the formation process of the native enzyme.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062562.

^* Corresponding author; e-mail kotake{at}molbiol.saitama-u.ac.jp; fax 81–48–858–3384.

Received March 9, 2005; returned for revision April 14, 2005; accepted April 17, 2005.

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