Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

Clemens Mayer*, Christopher Dulson, Eswar Reddem, Andy-Mark W. H. Thunnissen, Gerard Roelfes

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

60 Citaten (Scopus)
467 Downloads (Pure)


The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (k(cat)) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.

Originele taal-2English
Pagina's (van-tot)2083-2087
Aantal pagina's5
TijdschriftAngewandte Chemie-International Edition
Nummer van het tijdschrift7
StatusPublished - 11-feb.-2019


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