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Updated: Apr 9, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
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Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease
Thomas Shafee1,2,3, Pietro Gatti-Lafranconi1, Ralph Minter2
1Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA (UK).
Chembiochem : a European Journal of Chemical Biology
|June 23, 2015
Summary
Directed evolution restored activity to a mutated tobacco etch virus (TEV) protease. The evolved enzyme retained dual nucleophile capability, explaining evolutionary flexibility in protease superfamilies.
Area of Science:
- Enzymology and Protease Evolution
- Molecular Biology and Directed Evolution
Background:
- Mutation of the tobacco etch virus (TEV) protease nucleophile from cysteine to serine results in a significant loss of activity.
- Understanding the mechanisms and evolutionary pathways of enzyme adaptation is crucial in molecular biology.
Purpose of the Study:
- To investigate the effects of nucleophile mutation on TEV protease activity.
- To explore the evolutionary trajectory of TEV protease adapting to a serine nucleophile.
- To understand how enzymes can retain ancestral functions while adapting to new ones.
Main Methods:
- Directed evolution was employed over ten rounds to engineer the serine mutant (TEVSer).
- Enzyme activity assays were performed to compare wild-type, TEVSer, and evolved variants (TEVSerX).
- Comparative analysis of cysteine and serine nucleophile reactivity in evolved variants.
Main Results:
- Directed evolution recovered near-wild-type activity in the serine mutant (TEVSerX).
- Evolved variants retained the ability to utilize the ancestral cysteine nucleophile, indicating nucleophile permissiveness.
- Mutations enhancing serine nucleophile reactivity had minimal impact on cysteine nucleophile reactivity.
Conclusions:
- Adaptive evolution of TEVSer is paralleled by a neutral trajectory for TEV Cys, demonstrating evolutionary flexibility.
- Nucleophile permissiveness in evolved proteases explains how nucleophile switches can occur phylogenetically.
- Similar activities in evolved serine and cysteine variants may facilitate escape from adaptive conflict, enabling active-site evolution.
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