View source for 5tid


==X-ray structure of acyl-CoA thioesterase I, TesA, mutant M141L/Y145K/L146K at pH 5 in complex with octanoic acid==
<StructureSection load='5tid' size='340' side='right'caption='[[5tid]], [[Resolution|resolution]] 1.20&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[5tid]] is a 1 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=5TID OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5TID 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]] 1.2&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=OCA:OCTANOIC+ACID+(CAPRYLIC+ACID)'>OCA</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=5tid FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5tid OCA], [https://pdbe.org/5tid PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5tid RCSB], [https://www.ebi.ac.uk/pdbsum/5tid PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5tid ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/TESA_ECOLI TESA_ECOLI] Hydrolyzes only long chain acyl thioesters (C12-C18). Specificity similar to chymotrypsin.
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C6-C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of 'TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of 'TesA variants suggest that hydrophobic forces govern 'TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids.,Grisewood MJ, Hernandez Lozada NJ, Thoden JB, Gifford NP, Mendez-Perez D, Schoenberger HA, Allan MF, Floy ME, Lai RY, Holden HM, Pfleger BF, Maranas CD ACS Catal. 2017 Jun 2;7(6):3837-3849. doi: 10.1021/acscatal.7b00408. Epub 2017, Apr 20. PMID:29375928<ref>PMID:29375928</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 5tid" style="background-color:#fffaf0;"></div>

==See Also==
*[[Thioesterase 3D structures|Thioesterase 3D structures]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Escherichia coli]]
[[Category: Large Structures]]
[[Category: Allan MF]]
[[Category: Gifford NP]]
[[Category: Grisewood MJ]]
[[Category: Hernandez Lozada NJ]]
[[Category: Holden HM]]
[[Category: Marines CD]]
[[Category: Mendez-Perez D]]
[[Category: Pfleger BF]]
[[Category: Schoenberger HA]]
[[Category: Thoden JB]]