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PDBsum entry 1h2j

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Hydrolase PDB id
1h2j
Jmol
Contents
Protein chain
300 a.a. *
Ligands
DCB
GOL ×2
SO4
Waters ×378
* Residue conservation analysis
PDB id:
1h2j
Name: Hydrolase
Title: Endoglucanase cel5a in complex with unhydrolysed and covalently linked 2,4-dinitrophenyl-2-deoxy-2- fluoro-cellobioside at 1.15 a resolution
Structure: Endoglucanase 5a. Chain: a. Fragment: catalytic core domain only, residues 27-329. Synonym: endo-1,4-beta-glucanase, alkaline cellulase, cel5a. Engineered: yes
Source: Bacillus agaradhaerens. Organism_taxid: 76935. Strain: ac13. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.
Resolution:
1.15Å     R-factor:   0.118     R-free:   0.136
Authors: A.Varrot,G.J.Davies
Key ref:
A.Varrot and G.J.Davies (2003). Direct experimental observation of the hydrogen-bonding network of a glycosidase along its reaction coordinate revealed by atomic resolution analyses of endoglucanase Cel5A. Acta Crystallogr D Biol Crystallogr, 59, 447-452. PubMed id: 12595701 DOI: 10.1107/S0907444902023405
Date:
09-Aug-02     Release date:   15-Aug-02    
Supersedes: 1hf7
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
O85465  (GUN5_BACAG) -  Endoglucanase 5A
Seq:
Struc:
400 a.a.
300 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.4  - Cellulase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     carbohydrate metabolic process   1 term 
  Biochemical function     hydrolase activity, hydrolyzing O-glycosyl compounds     1 term  

 

 
DOI no: 10.1107/S0907444902023405 Acta Crystallogr D Biol Crystallogr 59:447-452 (2003)
PubMed id: 12595701  
 
 
Direct experimental observation of the hydrogen-bonding network of a glycosidase along its reaction coordinate revealed by atomic resolution analyses of endoglucanase Cel5A.
A.Varrot, G.J.Davies.
 
  ABSTRACT  
 
Non-covalent interactions between protein and ligand at the active centre of glycosidases play an enormous role in catalysis. Dissection of these hydrogen-bonding networks is not merely important for an understanding of enzymatic catalysis, but is also increasingly relevant for the design of transition-state mimics, whose tautomeric state, hydrogen-bonding interactions and protonation contribute to tight binding. Here, atomic resolution ( approximately 1 A) analysis of a series of complexes of the 34 kDa catalytic core domain of the Bacillus agaradhaerens endoglucanase Cel5A is presented. Cel5A is a 'retaining' endoglucanase which performs catalysis via the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. Previous medium-resolution analyses of a series of enzymatic snapshots has revealed conformational changes in the substrate along the reaction coordinate (Davies et al., 1998). Here, atomic resolution analyses of the series of complexes along the pathway are presented, including the 'Michaelis' complex of the unhydrolysed substrate, the covalent glycosyl-enzyme intermediate and the complex with the reaction product, cellotriose. These structures reveal intimate details of the protein-ligand interactions, including most of the carbohydrate-associated H atoms and the tautomeric state of crucial active-centre groups in the pH 5 orthorhombic crystal form and serve to illustrate the potential for atomic resolution analyses to inform strategies for enzyme inhibition.
 
  Selected figure(s)  
 
Figure 4.
Figure 4 (a) Observed F[obs] - F[calc] `difference' electron density for the trapped covalent 2-fluoro-2-deoxy-cellotriosyl-enzyme intermediate of Cel5A. The map, contoured at +4 , reveals the location of active-centre H atoms. Only density for the -1 and -2 subsites is shown for clarity. (b) Schematic figure of the hydrogen bonding within the Cel5A active centre. Experimentally determined H atoms are indicated in blue, those with weak density in green and those for which no electron density was observed in red.
Figure 5.
Figure 5 (a) Overlay of Cel5A structures along the reaction coordinate. Michaelis complex (plus the 35% of trapped intermediate) (blue), transition-state mimicking cellobio-imidazole (Varrot et al., 1999[Varrot, A., Schülein, M., Pipelier, M., Vasella, A. & Davies, G. J. (1999). J. Am. Chem. Soc. 121, 2621-2622.]) (yellow), covalent intermediate (red) and reaction product (green). A feature of these complexes is the static nature of the protein, with the exception of the nucleophile which rotates to avoid a steric clash with the 2-F substituent in the trapped 2-fluoro-glycosyl-enzyme intermediates. (b) Simplified schematic representation of the electrophilic migration of C1 along the reaction coordinate (with thanks to Dr David Vocadlo).
 
  The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2003, 59, 447-452) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19543714 H.Schagerlöf, C.Nilsson, L.Gorton, F.Tjerneld, H.Stålbrand, and A.Cohen (2009).
Use of 18O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase.
  Anal Bioanal Chem, 394, 1977-1984.  
19050861 L.Lin, X.Meng, P.Liu, Y.Hong, G.Wu, X.Huang, C.Li, J.Dong, L.Xiao, and Z.Liu (2009).
Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution.
  Appl Microbiol Biotechnol, 82, 671-679.  
19279191 R.Suzuki, Z.Fujimoto, S.Ito, S.Kawahara, S.Kaneko, K.Taira, T.Hasegawa, and A.Kuno (2009).
Crystallographic snapshots of an entire reaction cycle for a retaining xylanase from Streptomyces olivaceoviridis E-86.
  J Biochem, 146, 61-70.
PDB codes: 2d1z 2d20 2d22 2d23 2d24
19053460 P.Peralta-Yahya, B.T.Carter, H.Lin, H.Tao, and V.W.Cornish (2008).
High-throughput selection for cellulase catalysts using chemical complementation.
  J Am Chem Soc, 130, 17446-17452.  
17766382 J.Dechancie, F.R.Clemente, A.J.Smith, H.Gunaydin, Y.L.Zhao, X.Zhang, and K.N.Houk (2007).
How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme-inhibitor complexes? Implications for enzyme design.
  Protein Sci, 16, 1851-1866.  
16214343 A.Gutteridge, and J.M.Thornton (2005).
Understanding nature's catalytic toolkit.
  Trends Biochem Sci, 30, 622-629.  
15573375 E.P.Mitchell, C.Sabin, L.Snajdrová, M.Pokorná, S.Perret, C.Gautier, C.Hofr, N.Gilboa-Garber, J.Koca, M.Wimmerová, and A.Imberty (2005).
High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 A resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches.
  Proteins, 58, 735-746.
PDB code: 1uzv
15853815 J.Jänis, J.Hakanpää, N.Hakulinen, F.M.Ibatullin, A.Hoxha, P.J.Derrick, J.Rouvinen, and P.Vainiotalo (2005).
Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography.
  FEBS J, 272, 2317-2333.
PDB code: 1xnk
15062085 J.Allouch, W.Helbert, B.Henrissat, and M.Czjzek (2004).
Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose.
  Structure, 12, 623-632.
PDB code: 1urx
15604820 L.Hildén, and G.Johansson (2004).
Recent developments on cellulases and carbohydrate-binding modules with cellulose affinity.
  Biotechnol Lett, 26, 1683-1693.  
15148317 L.Verdoucq, J.Morinière, D.R.Bevan, A.Esen, A.Vasella, B.Henrissat, and M.Czjze (2004).
Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate.
  J Biol Chem, 279, 31796-31803.
PDB codes: 1v02 1v03 1v08
15356002 T.M.Gloster, J.M.Macdonald, C.A.Tarling, R.V.Stick, S.G.Withers, and G.J.Davies (2004).
Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases.
  J Biol Chem, 279, 49236-49242.
PDB codes: 1w3j 1w3k 1w3l
14517232 K.Hövel, D.Shallom, K.Niefind, V.Belakhov, G.Shoham, T.Baasov, Y.Shoham, and D.Schomburg (2003).
Crystal structure and snapshots along the reaction pathway of a family 51 alpha-L-arabinofuranosidase.
  EMBO J, 22, 4922-4932.
PDB codes: 1pz2 1pz3 1qw8 1qw9
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.