PDBsum entry 1w2p

Go to PDB code: 
protein ligands metals Protein-protein interface(s) links
Hydrolase PDB id
Protein chains
346 a.a. *
EDO ×3
_CA ×2
Waters ×896
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: The 3-dimensional structure of a xylanase (xyn10a) from cellvibrio japonicus
Structure: Endo-1,4-beta-xylanase a precursor. Chain: a, b. Fragment: catalytic domain, residues 265-611. Synonym: xylanase a, 1,4-beta-d-xylan xylanohydrolase a, xyla. Engineered: yes
Source: Cellvibrio japonicus. Organism_taxid: 155077. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: organism formerly known as pseudomonas fluorescens
1.45Å     R-factor:   0.129     R-free:   0.152
Authors: E.J.Taylor,F.Vincent,H.J.Gilbert,G.J.Davies
Key ref:
S.R.Andrews et al. (2004). The use of forced protein evolution to investigate and improve stability of family 10 xylanases. The production of Ca2+-independent stable xylanases. J Biol Chem, 279, 54369-54379. PubMed id: 15452124 DOI: 10.1074/jbc.M409044200
07-Jul-04     Release date:   30-Sep-04    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P14768  (XYNA_CELJU) -  Endo-1,4-beta-xylanase A
611 a.a.
346 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Endo-1,4-beta-xylanase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.
 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: 10.1074/jbc.M409044200 J Biol Chem 279:54369-54379 (2004)
PubMed id: 15452124  
The use of forced protein evolution to investigate and improve stability of family 10 xylanases. The production of Ca2+-independent stable xylanases.
S.R.Andrews, E.J.Taylor, G.Pell, F.Vincent, V.M.Ducros, G.J.Davies, J.H.Lakey, H.J.Gilbert.
Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.
  Selected figure(s)  
Figure 3.
FIG. 3. Schematic of the CjXyn10 derivatives. The capital letter numbered residues are in the original catalytic domain of CjXyn10A (G1 is the first enzyme in the catalytic domain and corresponds to Gly-265 in the full-length xylanase). The residues in lowercase are as follows. Gal residues (prefixed by gal-in the text) are encoded by the multiple cloning region of pUC19; cf residues (prefixed by cf in the text) are from CfXyn10A and are inserted between amino acids Asn-248 and Ser-278 of CjXyn10A to generate CfCjXyn10A.
Figure 7.
FIG. 7. Crystal structure of the thermostable mutants of CjXyn10A. Panel a shows orthogonal views of CjXyn10A with the three sites of improved stability indicated; the calcium binding loop, Ala-80, and the N- and C-terminal "contact" region where an inserted S-S bridge contributes to stability. Panel b reveals the structural basis of the A80T mutation. The introduced threonine both fills a void and, thus, makes van der Waals contacts with surrounding residues and allows a favorable hydrogen-bond (dotted yellow line) with Asp-123. This figure was drawn with PyMol (DeLano Scientific, San Carlos, CA) and is in divergent ("wall-eyed") stereo.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 54369-54379) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19854928 S.Anbarasan, J.Jänis, M.Paloheimo, M.Laitaoja, M.Vuolanto, J.Karimäki, P.Vainiotalo, M.Leisola, and O.Turunen (2010).
Effect of glycosylation and additional domains on the thermostability of a family 10 xylanase produced by Thermopolyspora flexuosa.
  Appl Environ Microbiol, 76, 356-360.  
17876824 R.Ruller, L.Deliberto, T.L.Ferreira, and R.J.Ward (2008).
Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes.
  Proteins, 70, 1280-1293.  
16741587 A.S.Rowan, and C.J.Hamilton (2006).
Recent developments in preparative enzymatic syntheses of carbohydrates.
  Nat Prod Rep, 23, 412-443.  
16450065 C.C.Lee, M.Smith, R.E.Kibblewhite-Accinelli, T.G.Williams, K.Wagschal, G.H.Robertson, and D.W.Wong (2006).
Isolation and characterization of a cold-active xylanase enzyme from Flavobacterium sp.
  Curr Microbiol, 52, 112-116.  
16532363 C.C.Lee, R.E.Kibblewhite-Accinelli, K.Wagschal, G.H.Robertson, and D.W.Wong (2006).
Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library.
  Extremophiles, 10, 295-300.  
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.