Structural basis and catalytic mechanism for the dual functional endo-beta-N-acetylglucosaminidase A.
Endo-beta-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man(3)GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues - E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as "gate-keepers". Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.
Yin
Jie
J
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Li
Lei
L
Shaw
Neil
N
Li
Yang
Y
Song
Jing Katherine
JK
Zhang
Wenpeng
W
Xia
Chengfeng
C
Zhang
Rongguang
R
Joachimiak
Andrzej
A
Zhang
Hou-Cheng
HC
Wang
Lai-Xi
LX
Liu
Zhi-Jie
ZJ
Wang
Peng
P
D055162:Biocatalysis; D002240:Carbohydrate Sequence; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase