3QZU

Crystal structure of Bacillus subtilis Lipase A 7-fold mutant; the outcome of directed evolution towards thermostability


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.223 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention.

Augustyniak, W.Brzezinska, A.A.Pijning, T.Wienk, H.Boelens, R.Dijkstra, B.W.Reetz, M.T.

(2012) Protein Sci 21: 487-497

  • DOI: https://doi.org/10.1002/pro.2031
  • Primary Citation of Related Structures:  
    3QZU

  • PubMed Abstract: 

    Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65 °C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.


  • Organizational Affiliation

    Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Lipase estA
A, B
181Bacillus subtilis subsp. subtilis str. 168Mutation(s): 7 
Gene Names: BSU02700estAliplipA
EC: 3.1.1.3
UniProt
Find proteins for P37957 (Bacillus subtilis (strain 168))
Explore P37957 
Go to UniProtKB:  P37957
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP37957
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
D [auth A],
I [auth B]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
GOL
Query on GOL

Download Ideal Coordinates CCD File 
E [auth A],
F [auth A],
G [auth A],
J [auth B],
K [auth B]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
C [auth A],
H [auth B]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.223 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 59.163α = 90
b = 45.511β = 101.1
c = 77.613γ = 90
Software Package:
Software NamePurpose
REFMACrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-02-08
    Type: Initial release
  • Version 1.1: 2012-04-18
    Changes: Database references
  • Version 1.2: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description