3QMM

Structure of 6B, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.89 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.194 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

In vitro evolved non-aggregating and thermostable lipase: structural and thermodynamic investigation

Kamal, M.Z.Ahmad, S.Molugu, T.R.Vijayalakshmi, A.Deshmukh, M.V.Sankaranarayanan, R.Rao, N.M.

(2011) J Mol Biol 413: 726-741

  • DOI: https://doi.org/10.1016/j.jmb.2011.09.002
  • Primary Citation of Related Structures:  
    3QMM

  • PubMed Abstract: 

    Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named "6B" using multiple rounds of in vitro evolution. T(m) and optimum activity temperature of 6B is 78 °C and 65 °C, respectively, which is ~22 °C and 30 °C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance.


  • Organizational Affiliation

    Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Hyderabad 500007, India.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Lipase estA
A, B
181Bacillus subtilisMutation(s): 12 
Gene Names: estA
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
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.89 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.194 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 37.481α = 90
b = 55.206β = 90
c = 137.064γ = 90
Software Package:
Software NamePurpose
SCALEPACKdata scaling
MOLREPphasing
CNSrefinement
PDB_EXTRACTdata extraction

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2011-10-05
    Type: Initial release
  • Version 1.1: 2013-06-19
    Changes: Database references
  • Version 1.2: 2023-11-01
    Changes: Data collection, Database references, Refinement description