View source for 3nq2


==Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution R2 3/5G==
<StructureSection load='3nq2' size='340' side='right'caption='[[3nq2]], [[Resolution|resolution]] 2.02&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[3nq2]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3NQ2 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3NQ2 FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.02&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=IMD:IMIDAZOLE'>IMD</scene></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=3nq2 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3nq2 OCA], [https://pdbe.org/3nq2 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3nq2 RCSB], [https://www.ebi.ac.uk/pdbsum/3nq2 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3nq2 ProSAT]</span></td></tr>
</table>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with &gt;200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in &gt;400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of &gt;5x10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.

Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution.,Khersonsky O, Rothlisberger D, Wollacott AM, Murphy P, Dym O, Albeck S, Kiss G, Houk KN, Baker D, Tawfik DS J Mol Biol. 2011 Apr 1;407(3):391-412. doi: 10.1016/j.jmb.2011.01.041. Epub 2011 , Jan 28. PMID:21277311<ref>PMID:21277311</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 3nq2" style="background-color:#fffaf0;"></div>

==See Also==
*[[Aldolase 3D structures|Aldolase 3D structures]]
*[[Kemp eliminase|Kemp eliminase]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Escherichia coli]]
[[Category: Large Structures]]
[[Category: Baker D]]
[[Category: Dym O]]
[[Category: Khersonsky O]]
[[Category: Rothlisberge D]]
[[Category: Tawfik DS]]
[[Category: Wollacott AM]]