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Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid
Author(s) -
Mayer Clemens,
Dulson Christopher,
Reddem Eswar,
Thunnissen AndyMark W. H.,
Roelfes Gerard
Publication year - 2019
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201813499
Subject(s) - aniline , enzyme , amino acid , chemistry , enzyme kinetics , residue (chemistry) , catalysis , amino acid residue , side chain , directed evolution , tyrosine , protein design , stereochemistry , protein engineering , combinatorial chemistry , biochemistry , active site , protein structure , peptide sequence , organic chemistry , mutant , gene , polymer
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.