PDBsum entry 3d2c

Hydrolase

PDB id: 3d2c

Contents

Protein chains

(+ 6 more) 179 a.a. *

Waters ×1130

* Residue conservation analysis

PDB id:

3d2c

Name:

Hydrolase

Title:

Structure of 4d3, a thermostable mutant of bacillus subtilis lipase obtained through directed evolution

Structure:

Lipase. Chain: a, b, c, d, e, f, g, h, i, j, k, l. Synonym: triacylglycerol lipase. Engineered: yes. Mutation: yes

Source:

Bacillus subtilis. Organism_taxid: 1423. Gene: lipa, lip, bsu02700. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.18Å R-factor: 0.202 R-free: 0.245

Authors:

R.Sankaranarayanan,M.Z.Kamal

Key ref:

S.Ahmad et al. (2008). Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight. J Mol Biol, 381, 324-340. PubMed id: 18599073 DOI: 10.1016/j.jmb.2008.05.063

Date:

08-May-08 Release date: 03-Jun-08

Protein chains

P37957  (ESTA_BACSU) -  Lipase EstA from Bacillus subtilis (strain 168)

Seq: 212 a.a.

Struc: 179 a.a.*

* PDB and UniProt seqs differ at 9 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.1.3 - triacylglycerol lipase.
• Reaction: a triacylglycerol + H2O = a diacylglycerol + a fatty acid + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2008.05.063

J Mol Biol 381:324-340 (2008)

PubMed id: 18599073

Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight.

S.Ahmad, M.Z.Kamal, R.Sankaranarayanan, N.M.Rao.

ABSTRACT

In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 degrees C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol(-1) in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 degrees C, the activity has increased two- to fivefold in the temperature range 25-65 degrees C. High-resolution crystal structures of three of the mutants, each with 5 degrees increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins.

Selected figure(s)

Figure 5.
Fig. 5. Ribbon diagram of the LipA molecule indicating the relative position of each mutation.

Figure 7.
Fig. 7. Structural changes in the regions around mutations identified in the third generation. (a) A15S mutation in the background of F17S mutation makes a hydrogen bond with the same. (b) Region around mutation A20E showing a water molecule making hydrogen bonds with side chains of residues Glu20, Ser24 and Arg33. (c) Electrostatic network consisting of side chains of Arg107 and Asp144, main-chain carbonyl groups of residues 107 and 142 and three water molecules named WT1, WT2 and WT3 in the wild-type protein. (d) Immediate vicinity around G111D mutation showing WT2 is replaced by carboxylate oxygen of Asp111 in the mutant. Stick representations of loop regions 107–108 and 142–143 are overlaid on the ribbon representations. In all the figures, water molecules are shown as red spheres and small blue spheres show electrostatic interactions.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 381, 324-340) copyright 2008.

Figures were selected by an automated process.