Wilkinson, HC;
Dalby, PA;
(2020)
Fine-tuning the activity and stability of an evolved enzyme active-site through non-canonical amino-acids.
The FEBS Journal
10.1111/febs.15560.
(In press).
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Abstract
Site‐specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade‐off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild‐type. The aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para‐aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para‐cyanophenylalanine (pCNF) and para‐nitrophenylalanine (pNTF), and simultaneously modified the hydrogen‐bonding potential of the aromatic substituent from accepting to donating. The fine‐tuning of residue 385 yielded variants with a 43‐fold increase in specific activity for 50 mm 3‐HBA and 100% increased kcat (pCNF), 290% improvement in Km (pNTF), 240% improvement in kcat/Km (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring‐substituted functional group, from donating to accepting, stabilised a helix‐turn (D259‐H261) through an intersubunit H‐bond with G262, to give a 7.8 °C increase in the thermal transition mid‐point, Tm, and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site‐specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs.
| Type: | Article |
|---|---|
| Title: | Fine-tuning the activity and stability of an evolved enzyme active-site through non-canonical amino-acids |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1111/febs.15560 |
| Publisher version: | https://febs.onlinelibrary.wiley.com/journal/17424... |
| Language: | English |
| Additional information: | © 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
| 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 Engineering Science > Dept of Chemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10109814 |
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