Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues
- Victoria University of Wellington, New Zealand
- University of British Columbia, Canada
Abstract
Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Katherine J Robins
Funding
Royal Society of New Zealand (15-VUW-037)
- Wayne M Patrick
- David F Ackerley
Cancer Society of New Zealand (18.05)
- Mark J Calcott
- David F Ackerley
Royal Society of New Zealand (19-VUW-076)
- Wayne M Patrick
- David F Ackerley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Hall et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues
eLife 9:e59081.
https://doi.org/10.7554/eLife.59081
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