3QZU

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

Summary for 3QZU

• Entry DOI: 10.2210/pdb3qzu/pdb
• Related: 1I6W
• Descriptor: Lipase estA, CHLORIDE ION, SULFATE ION, ... (5 entities in total)
• Functional Keywords: alpha/beta hydrolase, hydrolase
• Biological source: Bacillus subtilis subsp. subtilis
• Cellular location: Secreted: P37957
• Total number of polymer chains: 2
• Total formula weight: 39256.50

Authors

Pijning, T.,Augustyniak, W.,Reetz, M.T.,Dijkstra, B.W. (deposition date: 2011-03-07, release date: 2012-02-08, Last modification date: 2024-11-06)

Primary citation

Augustyniak, W.,Brzezinska, A.A.,Pijning, T.,Wienk, H.,Boelens, R.,Dijkstra, B.W.,Reetz, M.T.
Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention.
Protein Sci., 21:487-497, 2012

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.

PubMed: 22267088
DOI: 10.1002/pro.2031

Experimental method

X-RAY DIFFRACTION (1.85 Å)