Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection.
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.
Dröge
Melloney J
MJ
Dept. of Pharmaceutical Biology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Boersma
Ykelien L
YL
van Pouderoyen
Gertie
G
Vrenken
Titia E
TE
Rüggeberg
Carsten J
CJ
Reetz
Manfred T
MT
Dijkstra
Bauke W
BW
Quax
Wim J
WJ
D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D019151:Peptide Library; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C079999:LipA protein, Bacteria; D019151:Peptide Library; D010710:Phosphates