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Journal of Biochemistry Advance Access published online on June 13, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm124
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© 2007 The Japanese Biochemical Society

Identification of the catalytic acid-base residue of Arthrobacter endo-ß-N-acetylglucosaminidase by chemical rescue of an inactive mutant

Kiyotaka Fujita1,*, Reiko Sato2, Kazunori Toma2, Kanefumi Kitahara1, Toshihiko Suganuma1, Kenji Yamamoto3 and Kaoru Takegawa4

1Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. 2The Noguchi Institute, 1-8-1, Kaga, Itabashi, Tokyo 173-0003, Japan. 3Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. 4Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

*To whom correspondence should be addressed: Dr. Kiyotaka Fujita: Tel. +81-99-285-8639, Fax: +81-99-285-8639, E-mail: kfujita{at}ms.kagoshima-u.ac.jp

Received April 25, 2007; Accepted May 26, 2007


  Abstract

Arthrobacter endo-ß-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyzes the hydrolysis and transglycosylation of asparagine-linked oligosaccharides of glycoproteins with retention of anomeric configuration. Glu-173 of Endo-A is a catalytically essential amino acid residue, and the corresponding residue is conserved in all GH family 85 members. The catalytic activity of Endo-A E173A mutant was rescued by the addition of sodium azide or sodium formate. Furthermore, the produced ß-glycosyl azide (Man5GlcNAc-ß-N3) retained the anomeric configuration, indicating that Glu-173 is the catalytic acid-base residue of Endo-A. This is the first identification of the catalytic residue for GH family 85 endo-ß-N-acetylglucosaminidases.

Key Words: azide, catalytic acid-base residue, chemical rescue, endo-ß-N-acetylglucosaminidase, inactive mutant


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