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Hydrolase PDB id
2vtf
Jmol
Contents
Protein chains
616 a.a. *
Ligands
B3P ×4
PGE ×4
Waters ×1500
* Residue conservation analysis
PDB id:
2vtf
Name: Hydrolase
Title: X-ray crystal structure of the endo-beta-n- acetylglucosaminidase from arthrobacter protophormiae e173q mutant reveals a tim barrel catalytic domain and two ancillary domains
Structure: Endo-beta-n-acetylglucosaminidase. Chain: a, b. Fragment: residues 25-645. Engineered: yes. Mutation: yes
Source: Arthrobacter protophormiae. Organism_taxid: 37930. Strain: aku0647. Expressed in: escherichia coli. Expression_system_taxid: 469008.
Resolution:
1.79Å     R-factor:   0.166     R-free:   0.202
Authors: Z.Ling,R.J.Bingham,M.D.L.Suits,J.W.B.Moir,A.J.Fairbanks, E.J
Key ref:
Z.Ling et al. (2009). The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation activity. J Mol Biol, 389, 1-9. PubMed id: 19327363 DOI: 10.1016/j.jmb.2009.03.050
Date:
14-May-08     Release date:   31-Mar-09    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
Q9ZB22  (Q9ZB22_9MICC) -  Endo-beta-N-acetylglucosaminidase
Seq:
Struc:
 
Seq:
Struc:
645 a.a.
616 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.96  - Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of the di-N-acetylchitobiosyl unit in high-mannose glycopeptides and glycoproteins containing the -[Man(GlcNAc)2]Asn- structure. One N-acetyl-D-glucosamine residue remains attached to the protein; the rest of the oligosaccharide is released intact.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biological process     metabolic process   1 term 
  Biochemical function     hydrolase activity     4 terms  

 

 
DOI no: 10.1016/j.jmb.2009.03.050 J Mol Biol 389:1-9 (2009)
PubMed id: 19327363  
 
 
The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation activity.
Z.Ling, M.D.Suits, R.J.Bingham, N.C.Bruce, G.J.Davies, A.J.Fairbanks, J.W.Moir, E.J.Taylor.
 
  ABSTRACT  
 
Glycoside hydrolase family GH85 is a family of endo-beta-N-acetylglucosaminidases that is responsible for the hydrolysis of beta-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 A resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (beta/alpha)(8) catalytic domain occurs with two ancillary beta-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.
 
  Selected figure(s)  
 
Figure 1.
Fig. 1. The reaction and roles of Endo-A. (a) The reaction catalyzed by GH85 enzymes is the hydrolysis of the chitobiosyl core of N-linked glycans. (b) Neighboring-group participation mechanism for Endo-A via an oxazoline intermediate. (c) The synthetic utility of Endo-A and its variants in harnessing N-glycan oxazoline donors for the synthesis of glycoproteins with defined glycoforms.
Figure 3.
Fig. 3. Mechanistic questions raised by asparagine at position 171. (a) Reaction via a charged oxazolinium-ion-like intermediate in which the carbonyl of N171 aids the formation of the intermediate. (b) Reaction via an uncharged oxazoline intermediate that demands N171 to act as a base, perhaps via its imine tautomer.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 389, 1-9) copyright 2009.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19880511 M.Umekawa, C.Li, T.Higashiyama, W.Huang, H.Ashida, K.Yamamoto, and L.X.Wang (2010).
Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.
  J Biol Chem, 285, 511-521.  
20449490 T.B.Parsons, M.K.Patel, A.B.Boraston, D.J.Vocadlo, and A.J.Fairbanks (2010).
Streptococcus pneumoniae endohexosaminidase D; feasibility of using N-glycan oxazoline donors for synthetic glycosylation of a GlcNAc-asparagine acceptor.
  Org Biomol Chem, 8, 1861-1869.  
20396401 T.M.Gloster, and D.J.Vocadlo (2010).
Mechanism, Structure, and Inhibition of O-GlcNAc Processing Enzymes.
  Curr Signal Transduct Ther, 5, 74-91.  
20552664 T.V.Vuong, and D.B.Wilson (2010).
Glycoside hydrolases: catalytic base/nucleophile diversity.
  Biotechnol Bioeng, 107, 195-205.  
20486148 W.Huang, Q.Yang, M.Umekawa, K.Yamamoto, and L.X.Wang (2010).
Arthrobacter endo-beta-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C.
  Chembiochem, 11, 1350-1355.  
19766528 L.X.Wang, and W.Huang (2009).
Enzymatic transglycosylation for glycoconjugate synthesis.
  Curr Opin Chem Biol, 13, 592-600.  
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