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Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues

journal contribution
posted on 2021-02-24, 22:43 authored by KR Hall, KJ Robins, EM Williams, MH Rich, Mark CalcottMark Calcott, JN Copp, RF Little, Ralf SchwoererRalf Schwoerer, Gary EvansGary Evans, Wayne PatrickWayne Patrick, David AckerleyDavid Ackerley
© 2020, eLife Sciences Publications Ltd. All rights reserved. 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.


Better, Faster, Stronger: Bionic Enzymes for Artificial Substrates | Funder: ROYAL SOCIETY OF NEW ZEALAND | Grant ID: 15-VUW-037

Molecular Contingency on a Massive Scale: How Entirely New Antibiotic Resistance Genes Evolve | Funder: ROYAL SOCIETY OF NEW ZEALAND | Grant ID: 19-VUW-076


Preferred citation

Hall, K. R., Robins, K. J., Williams, E. M., Rich, M. H., Calcott, M. J., Copp, J. N., Little, R. F., Schwörer, R., Evans, G. B., Patrick, W. M. & Ackerley, D. F. (2020). Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active site residues. eLife, 9, 1-40.

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eLife Sciences Publications, Ltd

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