1R4Z

Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor

1R4Z の概要

• エントリーDOI: 10.2210/pdb1r4z/pdb
• 関連するPDBエントリー: 1I6W 1R50
• 分子名称: Lipase, [(4R)-2,2-DIMETHYL-1,3-DIOXOLAN-4-YL]METHYL HYDROGEN HEX-5-ENYLPHOSPHONATE (3 entities in total)
• 機能のキーワード: lipase, alpha/beta hydrolase, phosphonate inhibitor, hydrolase
• 由来する生物種: Bacillus subtilis
• 細胞内の位置: Secreted: P37957
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 39322.24

構造登録者

Droege, M.J.,Van Pouderoyen, G.,Vrenken, T.E.,Rueggeberg, C.J.,Reetz, M.T.,Dijkstra, B.W.,Quax, W.J. (登録日: 2003-10-09, 公開日: 2004-10-19, 最終更新日: 2024-10-30)

主引用文献

Droege, M.J.,Boersma, Y.L.,van Pouderoyen, G.,Vrenken, T.E.,Rueggeberg, C.J.,Reetz, M.T.,Dijkstra, B.W.,Quax, W.J.
Directed Evolution of Bacillus subtilis Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection
Chembiochem, 7:149-157, 2005

PubMed 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.

PubMed: 16342303
DOI: 10.1002/cbic.200500308

実験手法

X-RAY DIFFRACTION (1.8 Å)