Crystal Structure of GH101 Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

• Published in: Journal of Applied Glycoscience Vol. 56; no. 2; pp. 105 - 110
• Main Authors: Suzuki, Ryuichiro, Katayama, Takane, Fushinobu, Shinya, Kitaoka, Motomitsu, Kumagai, Hidehiko, Wakagi, Takayoshi, Shoun, Hirofumi, Ashida, Hisashi, Yamamoto, Kenji
• Format: Journal Article
• Language: Japanese
• Published: The Japanese Society of Applied Glycoscience 2009
• Subjects: Bifidobacteria; core 1 disaccharide; endo-α-N-acetyl galactosaminidase; galacto-N-biose, mucin-type O-glycan; glycoside hydrolase family 101
• ISSN: 1344-7882; 1880-7291
• DOI: 10.5458/jag.56.105

Endo-α-N-acetylgalactosaminidase (endo-α), a member of glycoside hydrolase (GH) family 101, catalyzes the hydrolysis of O-glycosidic α linkages of mucin-type O-glycan. Endo-α can be used in the synthesis of various glycoconjugates because of its transglycosylation activity. Therefore, it is important to elucidate the structure-function relationship of this enzyme. The gene encoding endo-α from Bifidobacterium longum JCM1217 has been cloned, and its gene product (EngBF) has been characterized in detail. EngBF releases a galacto-N-biose (Galβ1-3GalNAc, GNB) from glycoconjugates without damaging either the glycan or the core protein. This study presents the crystal structure of EngBF at 2.25 Å resolution. The catalytic domain of EngBF resembles the TIM barrel fold of GH13 α-amylase family. Based on structural comparison with α-amylase family, the catalytic nucleophile and acid/base catalyst residues of EngBF are determined to be Asp789 and Glu822, respectively. Moreover, the structural basis of substrate recognition by EngBF was predicted by automated docking and mutational studies, and was compared with endo-α from Clostridium perfringens strain 13 (EngCP). 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. Results of our present study provide insights into both the reaction and substrate recognition mechanisms of endoglycosidases that liberate mucin-type O-glycan from glycoconjugates.