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PDBsum entry 1h41

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protein ligands metals Protein-protein interface(s) links
Hydrolase PDB id
1h41
Jmol
Contents
Protein chains
700 a.a. *
Ligands
GCV ×2
EDO ×28
Metals
_CO ×10
Waters ×1667
* Residue conservation analysis
PDB id:
1h41
Name: Hydrolase
Title: Pseudomonas cellulosa e292a alpha-d-glucuronidase mutant complexed with aldotriuronic acid
Structure: Alpha-glucuronidase. Chain: a, b. Engineered: yes. Mutation: yes
Source: Pseudomonas cellulosa. Organism_taxid: 155077. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
Resolution:
1.5Å     R-factor:   0.118     R-free:   0.153
Authors: D.Nurizzo,T.Nagy,H.J.Gilbert,G.J.Davies
Key ref:
T.Nagy et al. (2003). The alpha-glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants. J Biol Chem, 278, 20286-20292. PubMed id: 12654910 DOI: 10.1074/jbc.M302205200
Date:
25-Sep-02     Release date:   01-May-03    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
B3PC73  (B3PC73_CELJU) -  Extracellular xylan exo-alpha-(1->2)-glucuronosidase
Seq:
Struc:
 
Seq:
Struc:
732 a.a.
700 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.131  - Xylan alpha-1,2-glucuronosidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of alpha-D-1,2-(4-O-methyl)glucuronosyl links in the main chain of hardwood xylans.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   3 terms 
  Biological process     metabolic process   5 terms 
  Biochemical function     hydrolase activity     4 terms  

 

 
DOI no: 10.1074/jbc.M302205200 J Biol Chem 278:20286-20292 (2003)
PubMed id: 12654910  
 
 
The alpha-glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants.
T.Nagy, D.Nurizzo, G.J.Davies, P.Biely, J.H.Lakey, D.N.Bolam, H.J.Gilbert.
 
  ABSTRACT  
 
alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.
 
  Selected figure(s)  
 
Figure 2.
FIG. 2. Schematic representation of the recognition elements in 4-O-MeGlcA binding derived from the GlcA67A E292A mutant in complex with aldobiouroniuc acid. Panel a displays the interactions between GlcA67A and 4-O-MeGlcA, whereas panel b displays the orientation of 4-O-MeGlcA and GlcA, respectively, within the active site of the -glucuronidase.
Figure 5.
FIG. 5. Circular dichroism spectroscopy of native and mutant forms on GlcA67A. Native (green) and mutants W160A (blue), V210S (black), V210N (red), K288A (pink), R325A (green dashed), K360A (red dashed), and W543A (blue dashed) were subjected to circular dichroism spectroscopy as described under "Experimental Procedures."
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 20286-20292) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
17921311 V.Chow, G.Nong, and J.F.Preston (2007).
Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2.
  J Bacteriol, 189, 8863-8870.  
17028274 F.J.St John, J.D.Rice, and J.F.Preston (2006).
Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan.
  J Bacteriol, 188, 8617-8626.  
15466046 D.Shallom, G.Golan, G.Shoham, and Y.Shoham (2004).
Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.
  J Bacteriol, 186, 6928-6937.  
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