6I8N

Crystal structure of LmrR with V15 replaced by unnatural amino acid 4-amino-L-phenylalanine

Summary for 6I8N

• Entry DOI: 10.2210/pdb6i8n/pdb
• Descriptor: Transcriptional regulator, PadR-like family, 3[N-MORPHOLINO]PROPANE SULFONIC ACID (3 entities in total)
• Functional Keywords: padr family, transcriptional regulator, engineered variant, artificial enzyme, unnatural amino acid, dna binding protein
• Biological source: Lactococcus lactis subsp. cremoris (strain MG1363)
• Total number of polymer chains: 2
• Total formula weight: 29864.95

Authors

Reddem, R.,Thunnissen, A.M.W.H. (deposition date: 2018-11-20, release date: 2019-01-02, Last modification date: 2024-01-24)

Primary citation

Mayer, C.,Dulson, C.,Reddem, E.,Thunnissen, A.W.H.,Roelfes, G.
Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid.
Angew. Chem. Int. Ed. Engl., 58:2083-2087, 2019

PubMed 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 ) 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.

PubMed: 30575260
DOI: 10.1002/anie.201813499

Experimental method

X-RAY DIFFRACTION (1.79 Å)