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Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

Yu, H; López, RIH; Steadman, D; Méndez-Sánchez, D; Higson, S; Cázares-Körner, A; Sheppard, TD; ... Dalby, PA; + view all (2020) Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones. The FEBS Journal , 287 (9) pp. 1758-1776. 10.1111/febs.15108. Green open access

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Abstract

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C2‐ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3‐formylbenzoic acid (FBA). Twenty single‐mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2‐fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild‐type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3‐FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high‐value products.

Type: Article
Title: Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1111/febs.15108
Publisher version: https://doi.org/10.1111/febs.15108
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: docking, protein engineering, rational design, transketolase, two substrates specificity
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases
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/10084395
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