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| | ==Crystal structure of dienelactone hydrolase (C123S) at 1.70 A resolution== | | ==Crystal structure of dienelactone hydrolase (C123S) at 1.70 A resolution== |
| - | <StructureSection load='4u2b' size='340' side='right' caption='[[4u2b]], [[Resolution|resolution]] 1.70Å' scene=''> | + | <StructureSection load='4u2b' size='340' side='right'caption='[[4u2b]], [[Resolution|resolution]] 1.70Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[4u2b]] is a 1 chain structure with sequence from [http://en.wikipedia.org/wiki/Dsm_6978 Dsm 6978]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4U2B OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4U2B FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[4u2b]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_knackmussii Pseudomonas knackmussii]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4U2B OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=4U2B FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.7Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4u2c|4u2c]], [[4u2d|4u2d]], [[4u2e|4u2e]], [[4u2f|4u2f]], [[4u2g|4u2g]]</td></tr>
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">clcD ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=65741 DSM 6978])</td></tr> | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=4u2b FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4u2b OCA], [https://pdbe.org/4u2b PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=4u2b RCSB], [https://www.ebi.ac.uk/pdbsum/4u2b PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=4u2b ProSAT]</span></td></tr> |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Carboxymethylenebutenolidase Carboxymethylenebutenolidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.45 3.1.1.45] </span></td></tr>
| + | |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4u2b FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4u2b OCA], [http://pdbe.org/4u2b PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4u2b RCSB], [http://www.ebi.ac.uk/pdbsum/4u2b PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4u2b ProSAT]</span></td></tr> | + | |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/CLCD_PSESB CLCD_PSESB]] Ring cleavage of cyclic ester dienelactone to produce maleylacetate. | + | [https://www.uniprot.org/uniprot/CLCD_PSEKB CLCD_PSEKB] Ring cleavage of cyclic ester dienelactone to produce maleylacetate. |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Carboxymethylenebutenolidase]] | + | [[Category: Large Structures]] |
| - | [[Category: Dsm 6978]] | + | [[Category: Pseudomonas knackmussii]] |
| - | [[Category: Collyer, C A]] | + | [[Category: Collyer CA]] |
| - | [[Category: Ollis, D L]] | + | [[Category: Ollis DL]] |
| - | [[Category: Porter, J L]] | + | [[Category: Porter JL]] |
| - | [[Category: A/b hydrolase fold]]
| + | |
| - | [[Category: Hydrolase]]
| + | |
| Structural highlights
Function
CLCD_PSEKB Ring cleavage of cyclic ester dienelactone to produce maleylacetate.
Publication Abstract from PubMed
The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse alpha/beta hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
Directed Evolution of New and Improved Enzyme Functions Using an Evolutionary Intermediate and Multidirectional Search.,Porter JL, Boon PL, Murray TP, Huber T, Collyer CA, Ollis DL ACS Chem Biol. 2014 Dec 5. PMID:25419863[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Porter JL, Boon PL, Murray TP, Huber T, Collyer CA, Ollis DL. Directed Evolution of New and Improved Enzyme Functions Using an Evolutionary Intermediate and Multidirectional Search. ACS Chem Biol. 2014 Dec 5. PMID:25419863 doi:http://dx.doi.org/10.1021/cb500809f
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