1r4z
From Proteopedia
(New page: 200px<br /><applet load="1r4z" size="450" color="white" frame="true" align="right" spinBox="true" caption="1r4z, resolution 1.80Å" /> '''Bacillus subtilis li...) |
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| - | [[Image:1r4z.jpg|left|200px]]<br /><applet load="1r4z" size=" | + | [[Image:1r4z.jpg|left|200px]]<br /><applet load="1r4z" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1r4z, resolution 1.80Å" /> | caption="1r4z, resolution 1.80Å" /> | ||
'''Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor'''<br /> | '''Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Phage display can be used as a protein-engineering tool for the 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1R4Z is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis] with RIL as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Triacylglycerol_lipase Triacylglycerol lipase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.3 3.1.1.3] Full crystallographic information is available from [http:// | + | 1R4Z is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis] with <scene name='pdbligand=RIL:'>RIL</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Triacylglycerol_lipase Triacylglycerol lipase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.3 3.1.1.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1R4Z OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Triacylglycerol lipase]] | [[Category: Triacylglycerol lipase]] | ||
| - | [[Category: Dijkstra, B | + | [[Category: Dijkstra, B W.]] |
| - | [[Category: Droege, M | + | [[Category: Droege, M J.]] |
| - | [[Category: Pouderoyen, G | + | [[Category: Pouderoyen, G Van.]] |
| - | [[Category: Quax, W | + | [[Category: Quax, W J.]] |
| - | [[Category: Reetz, M | + | [[Category: Reetz, M T.]] |
| - | [[Category: Rueggeberg, C | + | [[Category: Rueggeberg, C J.]] |
| - | [[Category: Vrenken, T | + | [[Category: Vrenken, T E.]] |
[[Category: RIL]] | [[Category: RIL]] | ||
[[Category: alpha/beta hydrolase]] | [[Category: alpha/beta hydrolase]] | ||
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[[Category: phosphonate inhibitor]] | [[Category: phosphonate inhibitor]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:47:09 2008'' |
Revision as of 12:47, 21 February 2008
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Bacillus subtilis lipase A with covalently bound Rc-IPG-phosphonate-inhibitor
Overview
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.
About this Structure
1R4Z is a Single protein structure of sequence from Bacillus subtilis with as ligand. Active as Triacylglycerol lipase, with EC number 3.1.1.3 Full crystallographic information is available from OCA.
Reference
Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection., Droge MJ, Boersma YL, van Pouderoyen G, Vrenken TE, Ruggeberg CJ, Reetz MT, Dijkstra BW, Quax WJ, Chembiochem. 2006 Jan;7(1):149-57. PMID:16342303
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