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

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

23 Citations (Scopus)
390 Downloads (Pure)

Abstract

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.

Original languageEnglish
Pages (from-to)2083-2087
Number of pages5
JournalAngewandte Chemie-International Edition
Volume58
Issue number7
DOIs
Publication statusPublished - 11-Feb-2019

Keywords

  • directed evolution
  • enzyme catalysis
  • enzyme design
  • hydrazones
  • organocatalysis
  • NUCLEOPHILIC CATALYSIS
  • HYDRAZONE
  • BIOCATALYSIS
  • MECHANISM
  • UNIVERSE

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