Chemists reversibly evolve enzyme | September 14, 2016 Issue - Vol. 94 Issue 37 | Chemical & Engineering News
Volume 94 Issue 37 | p. 7 | News of The Week
Issue Date: September 19, 2016 | Web Date: September 14, 2016

Chemists reversibly evolve enzyme

Demonstration shows that structural dynamics play an important role in evolving new enzyme functions
Department: Science & Technology
News Channels: Biological SCENE
Keywords: structural biology, directed evolution, enzymes
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The only substantial conformational changes between the phosphotriesterase (gray) and the arylesterase (blue) are in loops 4, 5, and 7. The spheres indicate the zinc ions in the active site.
Credit: Nat. Chem. Biol.
Superimposed ribbon structures of the starting and ending points of the directed evolution from a phosphotriesterase to an arylesterase.
 
The only substantial conformational changes between the phosphotriesterase (gray) and the arylesterase (blue) are in loops 4, 5, and 7. The spheres indicate the zinc ions in the active site.
Credit: Nat. Chem. Biol.

Protein motions and structural dynamics play an important role in the evolution of new enzyme functions, an international team reports.

To explore the importance of protein dynamics, Nobuhiko Tokuriki of the University of British Columbia, Colin J. Jackson of Australian National University, and coworkers started with an enzyme called a phosphotriesterase that normally hydrolyzes organophosphates. They then “evolved” this enzyme into an arylesterase, one that instead hydrolyzes aromatic esters, by doing multiple rounds of directed evolution. Finally, they turned around and evolved that arylesterase back into a phosphotriesterase, analyzing the crystal structures and dynamics of multiple proteins along both the forward and reverse evolutionary trajectories (Nat. Chem. Biol. 2016, DOI: 10.1038/nchembio.2175).

Undertaking the process in both directions helps elucidate the connection between protein sequence, structure, and function.

The team’s analysis revealed that major changes in enzyme activity could be achieved by mutating amino acids remote to the active site. “Despite huge changes in activities—several orders of magnitude—there was really only one mutation directly in the active site,” Jackson says. “We don’t necessarily need to completely change the active site to achieve very large changes in function.”

Instead, remote mutations change interactions such as salt bridges and hydrogen bonds significantly over the evolutionary pathways. Along the trajectories, active site mutations occurred, followed by other mutations that helped optimize the positioning of the residues in the new active sites. That optimization involved changes in the motions of some loops in the proteins.

Some of the enzymes that the researchers identified along the trajectories could catalyze both reactions equally well and better than some naturally occurring enzymes for the same reactions. This bifunctionality seems to stem from the proteins’ flexibility, Jackson says.

But there’s a selective pressure during the evolution process to freeze out unnecessary motions by reducing that flexibility. “The mobility isn’t necessarily a positive thing for catalysis,” Jackson says. As the enzymes become more specialized, they also become less flexible in some regions.

The new study successfully linked the evolution of enzyme function to changing protein dynamics, says Joelle Pelletier, a chemistry professor at the University of Montreal who uses directed evolution to study protein catalysis. “Some regions adopt new wiggle-room to test out conformations, later stabilizing the most productive conformations with further evolution, while another switches from dynamic to rigid. That fine-tuning allowed specialization for one function to the detriment of the other.”

The researchers hope that their findings will be incorporated into methods for rationally designing proteins. “The obvious challenge is to take what we think we have learned and try to apply it rationally,” Jackson says. “If we can’t make things rationally, we’re still missing something.”

 
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