PDBsum entry 1r4z

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protein ligands links
Hydrolase PDB id
Protein chains
179 a.a. *
RIL ×2
Waters ×290
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Bacillus subtilis lipase a with covalently bound rc-ipg- phosphonate-inhibitor
Structure: Lipase. Chain: a, b. Fragment: residues 1-181. Synonym: lipase a. Engineered: yes
Source: Bacillus subtilis. Organism_taxid: 1423. Gene: lipa. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.
1.80Å     R-factor:   0.177     R-free:   0.210
Authors: M.J.Droege,G.Van Pouderoyen,T.E.Vrenken,C.J.Rueggeberg, M.T.Reetz,B.W.Dijkstra,W.J.Quax
Key ref: M.J.Dröge et al. (2006). Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection. Chembiochem, 7, 149-157. PubMed id: 16342303 DOI: 10.1002/cbic.200500308
09-Oct-03     Release date:   19-Oct-04    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P37957  (ESTA_BACSU) -  Lipase EstA
212 a.a.
179 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Triacylglycerol lipase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Triacylglycerol + H2O = diacylglycerol + a carboxylate
+ H(2)O
= diacylglycerol
+ carboxylate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     lipid metabolic process   2 terms 
  Biochemical function     hydrolase activity     2 terms  


DOI no: 10.1002/cbic.200500308 Chembiochem 7:149-157 (2006)
PubMed id: 16342303  
Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection.
M.J.Dröge, Y.L.Boersma, G.van Pouderoyen, T.E.Vrenken, C.J.Rüggeberg, M.T.Reetz, B.W.Dijkstra, W.J.Quax.
Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.

Literature references that cite this PDB file's key reference

  PubMed id Reference
21381205 K.E.Cassimjee, R.Kourist, D.Lindberg, M.Wittrup Larsen, N.H.Thanh, M.Widersten, U.T.Bornscheuer, and P.Berglund (2011).
One-step enzyme extraction and immobilization for biocatalysis applications.
  Biotechnol J, 6, 463-469.  
21351219 Y.Jiang, K.L.Morley, J.D.Schrag, and R.J.Kazlauskas (2011).
Different active-site loop orientation in serine hydrolases versus acyltransferases.
  Chembiochem, 12, 768-776.
PDB code: 3ia2
18566980 N.Otte, M.Bocola, and W.Thiel (2009).
Force-field parameters for the simulation of tetrahedral intermediates of serine hydrolases.
  J Comput Chem, 30, 154-162.  
18383241 Y.L.Boersma, M.J.Dröge, A.M.van der Sloot, T.Pijning, R.H.Cool, B.W.Dijkstra, and W.J.Quax (2008).
A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A.
  Chembiochem, 9, 1110-1115.  
17446890 M.T.Reetz, and J.D.Carballeira (2007).
Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes.
  Nat Protoc, 2, 891-903.  
17458914 S.Becker, A.Michalczyk, S.Wilhelm, K.E.Jaeger, and H.Kolmar (2007).
Ultrahigh-throughput screening to identify E. coli cells expressing functionally active enzymes on their surface.
  Chembiochem, 8, 943-949.  
17448143 Y.L.Boersma, M.J.Dröge, and W.J.Quax (2007).
Selection strategies for improved biocatalysts.
  FEBS J, 274, 2181-2195.  
17183507 Z.Qian, C.J.Fields, Y.Yu, and S.Lutz (2007).
Recent progress in engineering alpha/beta hydrolase-fold family members.
  Biotechnol J, 2, 192-200.  
16647151 M.Konarzycka-Bessler, and K.E.Jaeger (2006).
Select the best: novel biocatalysts for industrial applications.
  Trends Biotechnol, 24, 248-250.  
17075931 M.T.Reetz, J.D.Carballeira, and A.Vogel (2006).
Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability.
  Angew Chem Int Ed Engl, 45, 7745-7751.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.