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Constrained evolution of a bispecific enzyme: lessons for biocatalyst design
E Sugrue1, C Scott2, C J Jackson1
1Research School of Chemistry, Australian National University, Canberra, Australia. colin.jackson@anu.edu.au.
Organic & Biomolecular Chemistry
|January 6, 2017
Summary
Intramolecular epistasis, or mutation interactions, significantly restricts enzyme evolution. Studying triazine hydrolase (TrzN) evolution revealed that only one of 24 paths to bispecificity was viable due to these complex genetic interactions.
Area of Science:
- Biochemistry and Molecular Biology
- Enzyme Engineering
- Evolutionary Biology
Background:
- Predicting mutation effects on protein stability and activity is crucial for protein design.
- Intramolecular epistasis, complex mutation interactions, can lead to unpredictable, non-additive effects, limiting evolutionary paths.
- Triazine hydrolase (TrzN) enzymes evolved to metabolize herbicides, offering a model for studying enzyme evolution.
Purpose of the Study:
- To quantitatively investigate all possible evolutionary trajectories from monofunctional to bispecific triazine hydrolase (TrzN) isoforms.
- To identify viable evolutionary paths and understand the role of intramolecular epistasis in shaping enzyme evolution.
- To analyze the impact of mutations on enzyme activity, stability, expression, and structure.
Main Methods:
- Systematic investigation of all 24 possible evolutionary trajectories from monofunctional to bispecific TrzN.
- Quantitative assessment of enzyme activity, stability, expression, and structural changes across trajectories.
- Analysis of epistatic interactions influencing catalytic residues, thermostability, and protein expression.
Main Results:
- Half of the investigated evolutionary trajectories were unviable due to inactive intermediate forms.
- Only one out of 24 trajectories showed consistent improvement towards bispecificity.
- The mutation Gln241 to Glu241 was identified as a critical first step in the most viable evolutionary path, significantly increasing ametryn activity.
Conclusions:
- Intramolecular epistasis creates significant bottlenecks in enzyme evolution, severely restricting viable evolutionary pathways.
- The order of mutations is critical for successful protein engineering and evolution, especially when aiming for enhanced or novel functions.
- Understanding epistatic interactions is essential for accurately predicting and guiding protein evolution for desired traits.
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