Issue Date: October 23, 2017 | Web Date: October 17, 2017
Evolving enzymes for nonnatural amino acids
Cells construct their proteins mainly from a collection of 20 canonical amino acids. To study the function of proteins or to give the biomolecules new abilities, biochemists have spent decades finding ways to get cells to move beyond those 20 and use nonnatural amino acids.
One method of incorporating nonnatural amino acids into proteins requires biological machinery that can recognize the amino acid and attach it to its corresponding transfer RNA molecule, which a cell’s ribosome then uses to synthesize proteins. A pyrrolysyl-tRNA synthetase (PylRS) from archaea is a favorite for this job because it can be adapted to accept many different nonnatural amino acids but doesn’t recognize the canonical ones.
Researchers now report a structure that helps explain why PylRS is so versatile (Nat. Chem. Biol. 2017, DOI: 10.1038/nchembio.2497). The team then used a protein evolution method with PylRS as a starting point to generate highly active and selective new synthetases (Nat. Chem. Biol. 2017, DOI: 10.1038/nchembio.2474). The research was a collaboration between Dieter Söll’s group at Yale University and David R. Liu’s group at Harvard University.
The X-ray crystal structure of PylRS shows that the way it binds a tRNA accommodates the small variable arm of the pyrrolysyl tRNA but not the larger variable arm of canonical tRNAs, explaining why PylRS doesn’t interact with tRNAs that code for canonical amino acids.
Liu’s group used PylRS in a protein evolution method called phage-assisted continuous evolution (PACE). The technique improved the efficiency of PylRS as much as 45-fold relative to the parent enzyme. When the researchers made the PACE-evolved mutations in other PylRS-derived synthetases, they improved the activity of those enzymes without going through evolution. PACE also evolved synthetases with altered amino acid specificity.
“The work presented here allows for aminoacyl-tRNA synthetase (aaRS) evolution over multiple generations on a practical timescale,” says Wenshe Liu, who studies protein evolution at Texas A&M University. “These reports suggest that this new method for aaRS evolution can yield aaRS mutants with considerable improvements in activity and specificity.”
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