View source for 7puo


==Structure of a fused 4-OT variant engineered for asymmetric Michael addition reactions==
<StructureSection load='7puo' size='340' side='right'caption='[[7puo]], [[Resolution|resolution]] 2.35&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[7puo]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7PUO OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7PUO 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]] 2.35&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</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=7puo FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7puo OCA], [https://pdbe.org/7puo PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7puo RCSB], [https://www.ebi.ac.uk/pdbsum/7puo PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7puo ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/4OT1_PSEPU 4OT1_PSEPU] Catalyzes the ketonization of 2-hydroxymuconate stereoselectively to yield 2-oxo-3-hexenedioate.<ref>PMID:1339435</ref> 
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180 degrees relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme.,Xu G, Kunzendorf A, Crotti M, Rozeboom HJ, Thunnissen AWH, Poelarends GJ Angew Chem Int Ed Engl. 2021 Dec 10:e202113970. doi: 10.1002/anie.202113970. PMID:34890491<ref>PMID:34890491</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 7puo" style="background-color:#fffaf0;"></div>
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Pseudomonas putida]]
[[Category: Poelarends GJ]]
[[Category: Rozeboom HJ]]
[[Category: Thunnissen AMWH]]