Identification of the catalytic acid base residue of arthrobacter endo-beta-N-acetylglucosaminidase by chemical rescue of an inactive mutant.
Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyses 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 beta-glycosyl azide (Man(5)GlcNAc-beta-N(3)) 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-beta-N-acetylglucosaminidases.
Fujita
Kiyotaka
K
Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima, Japan. kfujita@ms.kagoshima-u.ac.jp
Sato
Reiko
R
Toma
Kazunori
K
Kitahara
Kanefumi
K
Suganuma
Toshihiko
T
Yamamoto
Kenji
K
Takegawa
Kaoru
K
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation
D017931:DNA Primers; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase