PDBsum entry 2w92

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Hydrolase PDB id
Protein chain
636 a.a. *
Waters ×935
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Structure of a streptococcus pneumoniae family 85 glycoside hydrolase, endo-d, in complex with NAG-thiazoline.
Structure: Endo-beta-n-acetylglucosaminidase d. Chain: a. Fragment: catalytic module, residues 159-807. Synonym: endo-d catalytic module. Engineered: yes
Source: Streptococcus pneumoniae. Organism_taxid: 170187. Strain: tigr4. Expressed in: escherichia coli. Expression_system_taxid: 469008.
1.65Å     R-factor:   0.149     R-free:   0.187
Authors: D.W.Abbott,M.S.Macauley,D.J.Vocadlo,A.B.Boraston
Key ref:
D.W.Abbott et al. (2009). Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases. J Biol Chem, 284, 11676-11689. PubMed id: 19181667 DOI: 10.1074/jbc.M809663200
21-Jan-09     Release date:   27-Jan-09    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q93HW0  (Q93HW0_STRPN) -  Endo-beta-N-acetylglucosaminidase D
1646 a.a.
636 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biochemical function     mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase activity     1 term  


DOI no: 10.1074/jbc.M809663200 J Biol Chem 284:11676-11689 (2009)
PubMed id: 19181667  
Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases.
D.W.Abbott, M.S.Macauley, D.J.Vocadlo, A.B.Boraston.
Endo-beta-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K(d) = 28 microm) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.
  Selected figure(s)  
Figure 1.
The reaction catalyzed by GH85 endo-β-glucosaminidases including Endo-D and substrates and inhibitors used in this study. A, reaction catalyzed by GH85 enzymes cleaves the chitobiose core of N-glycans to generate a free N-glycan bearing a single GlcNAc residue at the terminus, and the liberated protein in which a single GlcNAc residue is N-linked to the protein. The glycosidic bond cleaved is indicated with an arrow. B, Structure of the oxazoline intermediate proposed for glycoside hydrolases using a substrate assisted catalytic mechanism. C, structures of the series of β-glucosaminide substrates used in this study that have varying degrees of fluorination in the acyl group. D, structure of NAG-thiazoline.
Figure 3.
The structure of SpGH85. A, divergent stereo schematic representation of the 1.4 Å crystal structure of the catalytic region of SpGH85. The N-terminal catalytic domain (yellow) is followed by the D1 domain (purple) and then the D2 domain (blue). Relevant active site residues are shown in green stick representation. B, divergent stereo surface representation of SpGH85 shown from the same perspective as in A. The bound NAG-thiazoline molecule from the NAG-thiazoline complex is shown for reference. The surface areas contributed by relevant active residues are shown in purple (Tyr-373), green (acid/base, Glu-337), and blue (catalytic asparagine, Asn-335). The arrows approximate the parts of the active site that may be occupied by a branched, high mannose substrate.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 11676-11689) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20624274 C.Frolet, M.Beniazza, L.Roux, B.Gallet, M.Noirclerc-Savoye, T.Vernet, and A.M.Di Guilmi (2010).
New adhesin functions of surface-exposed pneumococcal proteins.
  BMC Microbiol, 10, 190.  
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.  
20066263 T.M.Gloster, and G.J.Davies (2010).
Glycosidase inhibition: assessing mimicry of the transition state.
  Org Biomol Chem, 8, 305-320.  
20552664 T.V.Vuong, and D.B.Wilson (2010).
Glycoside hydrolases: catalytic base/nucleophile diversity.
  Biotechnol Bioeng, 107, 195-205.  
19692330 F.Sabbadin, R.Jackson, K.Haider, G.Tampi, J.P.Turkenburg, S.Hart, N.C.Bruce, and G.Grogan (2009).
The 1.5-A structure of XplA-heme, an unusual cytochrome P450 heme domain that catalyzes reductive biotransformation of royal demolition explosive.
  J Biol Chem, 284, 28467-28475.
PDB codes: 2wiv 2wiy
19766528 L.X.Wang, and W.Huang (2009).
Enzymatic transglycosylation for glycoconjugate synthesis.
  Curr Opin Chem Biol, 13, 592-600.  
19523117 T.V.Vuong, and D.B.Wilson (2009).
The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B.
  FEBS J, 276, 3837-3845.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.