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Journal of Biochemistry Advance Access originally published online on June 5, 2009
Journal of Biochemistry 2009 146(3):389-398; 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,*

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

*To whom correspondence should be addressed. Tel/Fax: +81+3-5841-5151, E-mail: 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, hydrolyses 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 the 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, galacto-N-biose, glycoside hydrolase family 101, mucin-type O-glycan

Abbreviations: CBM, carbohydrate-binding module; core 1-pNP, Galβ1-3GalNAc{alpha}1-pNP; core 2-pNP, Galβ1-3(GlcNAcβ1-6)GalNAc{alpha}1-pNP; core 3-pNP, GlcNAcβ1-3GalNAc{alpha}1-pNP; endo-{alpha}-GalNAc-ase, endo-{alpha}-N-acetylgalactosaminidase; EngBF, endo-{alpha}-GalNAc-ase from Bifidobacterium longum; EngCP, endo-{alpha}-GalNAc-ase from Clostridium perfringens; EngEF, endo-{alpha}-GalNAc-ase from Enterococcus faecalis; EngPA, endo-{alpha}-GalNAc-ase from Propionibacterium acnes; EngSP, endo-{alpha}-GalNAc-ase from Streptococcus pneumoniae R6; GH, glycoside hydrolase; GNB, galacto-N-biose (Galβ1-3GalNAc); LGA, Lamarckian genetic algorithm; LNB, lacto-N-biose I (Galβ1-3GlcNAc); MAD, multiple-wavelength anomalous dispersion; mGNB, 1-{alpha}-methyl-GNB; MPD, 2-methyl-2,4-pentanediol; RMSD, root mean square deviations


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