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==Crystal Structure of Kemp Eliminase HG4 with bound transition state analogue, 277K== | ==Crystal Structure of Kemp Eliminase HG4 with bound transition state analogue, 277K== | ||
<StructureSection load='5rgf' size='340' side='right'caption='[[5rgf]]' scene=''> | <StructureSection load='5rgf' size='340' side='right'caption='[[5rgf]], [[Resolution|resolution]] 1.46Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5RGF OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=5RGF FirstGlance]. <br> | <table><tr><td colspan='2'>[[5rgf]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Theau Theau]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5RGF OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=5RGF FirstGlance]. <br> | ||
</td></tr><tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=5rgf FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5rgf OCA], [http://pdbe.org/5rgf PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5rgf RCSB], [http://www.ebi.ac.uk/pdbsum/5rgf PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5rgf ProSAT]</span></td></tr> | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=6NT:6-NITROBENZOTRIAZOLE'>6NT</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> | ||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.8 3.2.1.8] </span></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=5rgf FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5rgf OCA], [http://pdbe.org/5rgf PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5rgf RCSB], [http://www.ebi.ac.uk/pdbsum/5rgf PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5rgf ProSAT]</span></td></tr> | |||
</table> | </table> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M(-1)s(-1)). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M(-1)s(-1)) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. | |||
Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.,Broom A, Rakotoharisoa RV, Thompson MC, Zarifi N, Nguyen E, Mukhametzhanov N, Liu L, Fraser JS, Chica RA Nat Commun. 2020 Sep 23;11(1):4808. doi: 10.1038/s41467-020-18619-x. PMID:32968058<ref>PMID:32968058</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 5rgf" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Kemp eliminase|Kemp eliminase]] | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Endo-1,4-beta-xylanase]] | |||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: Broom A]] | [[Category: Theau]] | ||
[[Category: Chica | [[Category: Broom, A]] | ||
[[Category: Fraser | [[Category: Chica, R A]] | ||
[[Category: Rakotoharisoa | [[Category: Fraser, J S]] | ||
[[Category: Thompson | [[Category: Rakotoharisoa, R V]] | ||
[[Category: Thompson, M C]] | |||
[[Category: Hydrolase]] | |||
Revision as of 10:59, 2 December 2020
Crystal Structure of Kemp Eliminase HG4 with bound transition state analogue, 277KCrystal Structure of Kemp Eliminase HG4 with bound transition state analogue, 277K
Structural highlights
Publication Abstract from PubMedThe creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M(-1)s(-1)). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M(-1)s(-1)) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.,Broom A, Rakotoharisoa RV, Thompson MC, Zarifi N, Nguyen E, Mukhametzhanov N, Liu L, Fraser JS, Chica RA Nat Commun. 2020 Sep 23;11(1):4808. doi: 10.1038/s41467-020-18619-x. PMID:32968058[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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