Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection

Chembiochem. 2006 Jan;7(1):149-57. doi: 10.1002/cbic.200500308.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / drug effects
  • Bacillus subtilis / enzymology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / drug effects
  • Bacterial Proteins / genetics
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • Directed Molecular Evolution / methods*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Hydrolysis
  • Models, Molecular
  • Molecular Structure
  • Mutation
  • Peptide Library*
  • Phosphates / chemistry
  • Phosphates / pharmacology*
  • Protein Conformation
  • Protein Structure, Tertiary
  • Stereoisomerism
  • Structure-Activity Relationship

Substances

  • Bacterial Proteins
  • Enzyme Inhibitors
  • LipA protein, Bacteria
  • Peptide Library
  • Phosphates