Bernardes Pinheiro, VB;
(2018)
E. coli surface display of streptavidin for directed evolution of an allylic deallylase.
Chemical Science
, 9
(24)
pp. 5383-5388.
10.1039/C8SC00484F.
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Abstract
Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M–K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.
Type: | Article |
---|---|
Title: | E. coli surface display of streptavidin for directed evolution of an allylic deallylase |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.1039/C8SC00484F |
Publisher version: | http://doi.org/10.1039/C8SC00484F |
Language: | English |
Additional information: | Copyright This journal is © The Royal Society of Chemistry 2018. Open Access Article. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. - With correction dated 28 June 2019. |
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 Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
URI: | https://discovery.ucl.ac.uk/id/eprint/10049875 |
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