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1r50

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(New page: 200px<br /><applet load="1r50" size="450" color="white" frame="true" align="right" spinBox="true" caption="1r50, resolution 1.45&Aring;" /> '''Bacillus subtilis li...)
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[[Image:1r50.jpg|left|200px]]<br /><applet load="1r50" size="450" color="white" frame="true" align="right" spinBox="true"
 
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caption="1r50, resolution 1.45&Aring;" />
 
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'''Bacillus subtilis lipase A with covalently bound Sc-IPG-phosphonate-inhibitor'''<br />
 
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==Overview==
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==Bacillus subtilis lipase A with covalently bound Sc-IPG-phosphonate-inhibitor==
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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.
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<StructureSection load='1r50' size='340' side='right'caption='[[1r50]], [[Resolution|resolution]] 1.45&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[1r50]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1R50 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1R50 FirstGlance]. <br>
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.45&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=SIL:[(4S)-2,2-DIMETHYL-1,3-DIOXOLAN-4-YL]METHYL+HYDROGEN+HEX-5-ENYLPHOSPHONATE'>SIL</scene></td></tr>
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1r50 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1r50 OCA], [https://pdbe.org/1r50 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1r50 RCSB], [https://www.ebi.ac.uk/pdbsum/1r50 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1r50 ProSAT]</span></td></tr>
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/ESTA_BACSU ESTA_BACSU] Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.<ref>PMID:8396026</ref>
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/r5/1r50_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1r50 ConSurf].
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<div style="clear:both"></div>
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<div style="background-color:#fffaf0;">
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== Publication Abstract from PubMed ==
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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.
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==About this Structure==
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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<ref>PMID:16342303</ref>
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1R50 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 SIL 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1R50 OCA].
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==Reference==
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From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
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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:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16342303 16342303]
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</div>
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[[Category: Bacillus subtilis]]
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<div class="pdbe-citations 1r50" style="background-color:#fffaf0;"></div>
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[[Category: Single protein]]
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[[Category: Triacylglycerol lipase]]
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[[Category: Dijkstra, B.W.]]
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[[Category: Droege, M.J.]]
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[[Category: Pouderoyen, G.Van.]]
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[[Category: Quax, W.J.]]
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[[Category: Reetz, M.T.]]
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[[Category: Rueggeberg, C.J.]]
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[[Category: Vrenken, T.E.]]
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[[Category: SIL]]
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[[Category: alpha/beta hydrolase]]
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[[Category: lipase]]
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[[Category: phosphonate inhibitor]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 01:15:58 2007''
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==See Also==
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*[[Lipase 3D Structures|Lipase 3D Structures]]
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== References ==
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<references/>
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__TOC__
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</StructureSection>
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[[Category: Bacillus subtilis]]
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[[Category: Large Structures]]
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[[Category: Dijkstra BW]]
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[[Category: Droege MJ]]
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[[Category: Quax WJ]]
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[[Category: Reetz MT]]
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[[Category: Rueggeberg CJ]]
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[[Category: Van Pouderoyen G]]
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[[Category: Vrenken TE]]

Current revision

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

PDB ID 1r50

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