title: Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease.
abstract: 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 TEVSeris 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.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA (UK).|Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge, CB21 6GH (UK).|Present address: La Trobe Institute of Molecular Sciences, La Trobe University, Science Drive, Melbourne, Victoria 3086 (Australia).
authors.forename: Thomas
authors.initials: T
authors.lastname: Shafee
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA (UK).
authors.forename: Pietro
authors.initials: P
authors.lastname: Gatti-Lafranconi
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge, CB21 6GH (UK).
authors.forename: Ralph
authors.initials: R
authors.lastname: Minter
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA (UK).
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
keywords: PA clan; directed evolution; nucleophilic catalysis; proteases; tobacco etch virus