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Journal of Biochemistry Advance Access first published online on June 5, 2009
This version published online on June 8, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp086
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Crystallographic and mutational analyses of substrate recognition of endo-{alpha}-N-acetylgalactosaminidase from Bifidobacterium longum

Ryuichiro Suzuki1, Takane Katayama2, Motomitsu Kitaoka3, Hidehiko Kumagai2, Takayoshi Wakagi1, Hirofumi Shoun1, Hisashi Ashida4, Kenji Yamamoto4 and Shinya Fushinobu1,*

1 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa 921-8836, Japan
3 Enzyme Laboratory, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
4 Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

*Corresponding author: Shinya Fushinobu, Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel./Fax: +81+3-5841-5151, E-mail address: asfushi{at}mail.ecc.u-tokyo.ac.jp

Received March 24, 2009; Accepted May 25, 2009


  Abstract

Endo-{alpha}-N-acetylgalactosaminidase (endo-{alpha}-GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyzes the O-glycosidic bonds in mucin-type O-glycan between {alpha}-GalNAc and Ser/Thr. Endo-{alpha}-GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Galβ1-3GalNAc (GNB), whereas endo-{alpha}-GalNAc-ase from Clostridium perfringens (EngCP) exhibits broader substrate specificity. We determined the crystal structure of EngBF at 2.0 Å resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the β-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to variations in amino acid sequences in the regions forming the substrate binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo-{alpha}-GalNAc-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates.

Key Words: Bifidobacteria, endo-{alpha}-N-acetylgalactosaminidase, glycoside hydrolase family 101, galacto-N-biose, mucin-type O-glycan


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