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Biological Chemistry

Nonnatural amino acid amps up enzyme efficiency

Directed evolution with noncanonical amino acid leads to enzyme with new mechanism and increased efficiency

by Celia Henry Arnaud
June 1, 2019 | A version of this story appeared in Volume 97, Issue 22


Reaction catalyzed by a methylhistidine-containing lab-evolved enzyme.
This reaction is catalyzed by an evolved enzyme containing a catalytic nonnatural amino acid.

The combination of protein design and directed evolution is a powerful way to create enzymes with new functions. Now, Anthony P. Green and coworkers at the University of Manchester have thrown a nonnatural amino acid into the mix. They started with a protein computationally designed to catalyze Morita-Baylis-Hillman carbon-carbon bond formation and transformed it into an enzyme that catalyzes ester hydrolysis (Nature 2019, DOI: 10.1038/s41586-019-1262-8). First, they replaced the catalytic histidine in the active site with a noncanonical methylhistidine. Then they subjected that new enzyme to multiple rounds of laboratory evolution with an assay based on fluorescein detection. The most active variant hydrolyzed fluorescein 2-phenylacetate 9,000 times as efficiently as free methylhistidine in solution and 2,800 times as efficiently as the organocatalysts dimethylamino­pyridine and N-methyl imidazole. Further rounds of evolution gave rise to an ­enantioselective enzyme that preferentially hydrolyzes one of the two mirror-image isomers of fluorescein 2-phenylpropanoate. Modifying the methylhistidine completely shut down the evolved enzymes, confirming it as the key catalytic amino acid. The ability to include nonnatural catalytic amino acids could expand the range of chemistries available in lab-evolved enzymes.


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