title: Structural basis and catalytic mechanism for the dual functional endo-beta-N-acetylglucosaminidase A.
abstract: 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.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yin
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Jing Katherine
authors.initials: JK
authors.lastname: Song
authors.forename: Wenpeng
authors.initials: W
authors.lastname: Zhang
authors.forename: Chengfeng
authors.initials: C
authors.lastname: Xia
authors.forename: Rongguang
authors.initials: R
authors.lastname: Zhang
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: Hou-Cheng
authors.initials: HC
authors.lastname: Zhang
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
mesh_terms: 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
chemical_list: D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase