3QMM

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

Summary for 3QMM

• Entry DOI: 10.2210/pdb3qmm/pdb
• Related: 1I6W 3D2B 3D2C
• Descriptor: Lipase estA (2 entities in total)
• Functional Keywords: lipase, alpha/beta hydrolase, stability, directed evolution, hydrolase
• Biological source: Bacillus subtilis
• Cellular location: Secreted: P37957
• Total number of polymer chains: 2
• Total formula weight: 39145.67

Authors

Sankaranarayanan, R.,Kamal, M.Z. (deposition date: 2011-02-04, release date: 2011-10-05, Last modification date: 2023-11-01)

Primary citation

Kamal, M.Z.,Ahmad, S.,Molugu, T.R.,Vijayalakshmi, A.,Deshmukh, M.V.,Sankaranarayanan, R.,Rao, N.M.
In vitro evolved non-aggregating and thermostable lipase: structural and thermodynamic investigation
J.Mol.Biol., 413:726-741, 2011

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.

PubMed: 21925508
DOI: 10.1016/j.jmb.2011.09.002

Experimental method

X-RAY DIFFRACTION (1.89 Å)