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Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease

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journal contribution
posted on 2021-01-06, 05:24 authored by Thomas Shafee, P Gatti-Lafranconi, R Minter, F Hollfelder
© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mutation of the tobacco etch virus (TEV) protease nucleophile from cysteine to serine causes an approximately 104-fold loss in activity. Ten rounds of directed evolution of the mutant, TEVSer, overcame the detrimental effects of nucleophile exchange to recover near-wild-type activity in the mutant TEVSerX. Rather than respecialising TEV to the new nucleophile, all the enzymes along the evolutionary trajectory also retained the ability to use the original cysteine nucleophile. Therefore the adaptive evolution of TEVSer is paralleled by a neutral trajectory for TEVCys, in which mutations that increase serine nucleophile reactivity hardly affect the reactivity of cysteine. This apparent nucleophile permissiveness explains how nucleophile switches can occur in the phylogeny of the chymotrypsin-like protease PA superfamily. Despite the changed key component of their chemical mechanisms, the evolved variants TEVSerX and TEVCysX have similar activities; this could potentially facilitate escape from adaptive conflict to enable active-site evolution.


We thank several colleagues, particularly Sean Devenish for insightful comments on the manuscript. We acknowledge financial support from the Biotechnology and Biological Sciences Research Council and MedImmune. F.H. is an ERC Starting Investigator.

Biotechnology and Biological Sciences Research Council



Publication Date



ChemBioChem: a European journal of chemical biology






4p. (p. 1866-1869)


Wiley-VCH Verlag



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