4K6K

Crystal structure of CALB mutant D223G from Candida antarctica


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.266 
  • R-Value Work: 0.235 
  • R-Value Observed: 0.236 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

Xie, Y.An, J.Yang, G.Wu, G.Zhang, Y.Cui, L.Feng, Y.

(2014) J Biol Chem 289: 7994-8006

  • DOI: https://doi.org/10.1074/jbc.M113.536045
  • Primary Citation of Related Structures:  
    4K5Q, 4K6G, 4K6H, 4K6K

  • PubMed Abstract: 

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.


  • Organizational Affiliation

    From the State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China and.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Lipase B
A, B
326Moesziomyces antarcticusMutation(s): 1 
EC: 3.1.1.3
UniProt
Find proteins for P41365 (Pseudozyma antarctica)
Explore P41365 
Go to UniProtKB:  P41365
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP41365
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.266 
  • R-Value Work: 0.235 
  • R-Value Observed: 0.236 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 46.708α = 90
b = 87.091β = 90
c = 138.87γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-01-29
    Type: Initial release
  • Version 1.1: 2014-05-07
    Changes: Database references
  • Version 1.2: 2017-11-15
    Changes: Refinement description