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PDBsum entry 3vzk

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protein ligands Protein-protein interface(s) links
Hydrolase PDB id
3vzk
Jmol
Contents
Protein chains
185 a.a.
Ligands
SO4 ×2
Waters ×486
PDB id:
3vzk
Name: Hydrolase
Title: Crystal structure of the bacillus circulans endo-beta-(1,4)- (bcx) n35e mutant
Structure: Endo-1,4-beta-xylanase. Chain: a, b. Synonym: xylanase, 1,4-beta-d-xylan xylanohydrolase. Engineered: yes. Mutation: yes
Source: Bacillus circulans. Organism_taxid: 1397. Gene: xlna. Expressed in: escherichia coli. Expression_system_taxid: 562
Resolution:
1.55Å     R-factor:   0.163     R-free:   0.197
Authors: M.L.Ludwiczek,I.D'Angelo,G.N.Yalloway,M.Okon,J.E.Nielsen, N.C.Strynadka,S.G.Withers,L.P.Mcintosh
Key ref: M.L.Ludwiczek et al. (2013). Strategies for modulating the pH-dependent activity of a family 11 glycoside hydrolase. Biochemistry, 52, 3138-3156. PubMed id: 23578322 DOI: 10.1021/bi400034m
Date:
14-Oct-12     Release date:   08-May-13    
PROCHECK
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 Headers
 References

Protein chains
Pfam   ArchSchema ?
P09850  (XYNA_BACCI) -  Endo-1,4-beta-xylanase
Seq:
Struc:
213 a.a.
185 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.3.2.1.8  - 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     metabolic process   4 terms 
  Biochemical function     hydrolase activity     4 terms  

 

 
DOI no: 10.1021/bi400034m Biochemistry 52:3138-3156 (2013)
PubMed id: 23578322  
 
 
Strategies for modulating the pH-dependent activity of a family 11 glycoside hydrolase.
M.L.Ludwiczek, I.D'Angelo, G.N.Yalloway, J.A.Brockerman, M.Okon, J.E.Nielsen, N.C.Strynadka, S.G.Withers, L.P.McIntosh.
 
  ABSTRACT  
 
The pH-dependent activity of wild-type Bacillus circulans xylanase (BcX) is set by the pKa values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these pKa values and thereby shift the pHopt at which BcX is optimally active. Altering the global charge of BcX through random succinylation had no significant effect. Mutation of residues near or within the active site of BcX, but not directly contacting the catalytic carboxyls, either had little effect or reduced its pHopt, primarily by lowering the apparent pKa value of Glu78. However, mutations causing the largest pKa changes also impaired activity. Although not found as a general acid/base in naturally occurring xylanases, substitution of Glu172 with a His lowered the pHopt of BcX from 5.6 to 4.7 while retaining 8% activity toward a xylobioside substrate. Mutation of Asn35, which contacts Glu172, to either His or Glu also led to a reduction in pHopt by ∼1.2 units. Detailed pKa measurements by NMR spectroscopy revealed that, despite the opposite charges of the introduced residues, both the N35H and N35E forms of BcX utilize a reverse protonation mechanism. In this mechanism, the pKa value of the general acid is lower than that of the nucleophile, and only a small population of enzyme is in a catalytically competent ionization state. However, overall activity is maintained due to the increased strength of the general acid. This study illustrates several routes for altering the pH-dependent properties of xylanases, while also providing valuable insights into complex protein electrostatics.