Hickman, TWP;
Baud, D;
Benhamou, L;
Hailes, HC;
Ward, JM;
(2020)
Characterisation of four hotdog-fold thioesterases for their implementation in a novel organic acid production system.
Applied Microbiology and Biotechnology
, 104
pp. 4397-4406.
10.1007/s00253-020-10519-w.
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Abstract
With increasing interest in the diverse properties of organic acids and their application in synthetic pathways, developing biological tools for producing known and novel organic acids would be very valuable. In such a system, organic acids may be activated as coenzyme A (CoA) esters, then modified by CoA-dependent enzymes, followed by CoA liberation by a broad-acting thioesterase. This study has focused on the identification of suitable thioesterases (TE) for utilisation in such a pathway. Four recombinant hotdog-fold TEs were screened with a range of CoA esters in order to identify a highly active, broad spectrum TE. The TesB-like TE, RpaL, from Rhodopseudomonas palustris was found to be able to use aromatic, alicyclic and both long and short aliphatic CoA esters. Size exclusion chromatography, revealed RpaL to be a monomer of fused hotdog domains, in contrast to the complex quaternary structures found with similar TesB-like TEs. Nonetheless, sequence alignments showed a conserved catalytic triad despite the variation in quaternary arrangement. Kinetic analysis revealed a preference towards short-branched chain CoA esters with the highest specificity towards DL-β-hydroxybutyryl CoA (1.6 × 104 M-1 s-1), which was found to decrease as the acyl chain became longer and more functionalised. Substrate inhibition was observed with the fatty acyl n-heptadecanoyl CoA at concentrations exceeding 0.3 mM; however, this was attributed to its micellar aggregation properties. As a result of the broad activity observed with RpaL, it is a strong candidate for implementation in CoA ester pathways to generate modified or novel organic acids.
| Type: | Article |
|---|---|
| Title: | Characterisation of four hotdog-fold thioesterases for their implementation in a novel organic acid production system |
| Location: | Germany |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1007/s00253-020-10519-w |
| Publisher version: | https://doi.org/10.1007/s00253-020-10519-w |
| Language: | English |
| Additional information: | © 2020 Springer Nature. This article is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). |
| Keywords: | CoA, Organic acid, Promiscuous, RpaL, Thioesterase |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10093919 |
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