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J. Biol. Chem., Vol. 280, Issue 45, 37415-37422, November 11, 2005

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Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo--N-acetylgalactosaminidase from Bifidobacterium longum^*

Kiyotaka Fujita1, Fusako Oura, Noriko Nagamine, Takane Katayama¶, Jun Hiratake||, Kanzo Sakata||, Hidehiko Kumagai¶, and Kenji Yamamoto

From the Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, the ^¶Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, the ^||Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, and the Core Research for Evolutional Science and Technology Program of Japan Science and Technology Corporation (CREST-JST), Kawaguchi, Saitama 332-0012, Japan

We found endo--N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl 1,3-N-acetyl-D-galactosamine (Gal1,3GalNAc) -linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590–1381 amino acid residues) exhibited 31–53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Gal1,3GalNAc disaccharide from Gal1,3GalNAc1pNP and asialofetuin, but did not release GalNAc, Gal1,3(GlcNAc1,6)GalNAc, GlcNAc1,3GalNAc, and Gal1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by ¹H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo--N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

Received for publication, June 24, 2005 , and in revised form, September 1, 2005.

^* This work was supported by Grants-in-aid for Scientific Research from Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technol-ogy Agency, Scientific Research (B), No. 14360055, from the Japan Society for the Promotion of Science, and by the Ajinomoto Research Fund. 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 nucleotide sequence of engBF has been submitted to the GenBank^TM/EBI Data Bank with accession number AY836679 [GenBank] .

¹ To whom correspondence should be addressed: Dept. of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. Tel.: 81-75-753-4298; Fax: 81-75-753-9228; E-mail: kfujita{at}lif.kyoto.u.ac.jp.

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