Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues
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 nit...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2020-11-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/59081 |
| _version_ | 1811180944477913088 |
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| author | Kelsi R Hall Katherine J Robins Elsie M Williams Michelle H Rich Mark J Calcott Janine N Copp Rory F Little Ralf Schwörer Gary B Evans Wayne M Patrick David F Ackerley |
| author_facet | Kelsi R Hall Katherine J Robins Elsie M Williams Michelle H Rich Mark J Calcott Janine N Copp Rory F Little Ralf Schwörer Gary B Evans Wayne M Patrick David F Ackerley |
| author_sort | Kelsi R Hall |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T09:11:50Z |
| format | Article |
| id | doaj.art-7322f1ca185a4740b5cf914fee329367 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-11T09:11:50Z |
| publishDate | 2020-11-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-7322f1ca185a4740b5cf914fee3293672022-12-22T04:32:29ZengeLife Sciences Publications LtdeLife2050-084X2020-11-01910.7554/eLife.59081Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residuesKelsi R Hall0Katherine J Robins1https://orcid.org/0000-0001-5049-4246Elsie M Williams2Michelle H Rich3https://orcid.org/0000-0003-4876-4029Mark J Calcott4https://orcid.org/0000-0002-7736-8095Janine N Copp5https://orcid.org/0000-0001-6690-0480Rory F Little6Ralf Schwörer7https://orcid.org/0000-0002-9352-6559Gary B Evans8Wayne M Patrick9https://orcid.org/0000-0002-2718-8053David F Ackerley10https://orcid.org/0000-0002-6188-9902School of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New ZealandCentre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand; Ferrier Institute, Victoria University of Wellington, Wellington, New ZealandCentre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand; Ferrier Institute, Victoria University of Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington, New ZealandSchool of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington, New ZealandSelection 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.https://elifesciences.org/articles/59081nitroreductaseenzyme promiscuitysubstrate competitiondirected evolutionenzyme evolution |
| spellingShingle | Kelsi R Hall Katherine J Robins Elsie M Williams Michelle H Rich Mark J Calcott Janine N Copp Rory F Little Ralf Schwörer Gary B Evans Wayne M Patrick David F Ackerley Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues eLife nitroreductase enzyme promiscuity substrate competition directed evolution enzyme evolution |
| title | Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues |
| title_full | Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues |
| title_fullStr | Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues |
| title_full_unstemmed | Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues |
| title_short | Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues |
| title_sort | intracellular complexities of acquiring a new enzymatic function revealed by mass randomisation of active site residues |
| topic | nitroreductase enzyme promiscuity substrate competition directed evolution enzyme evolution |
| url | https://elifesciences.org/articles/59081 |
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