Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes

Area of Science:

• Protein evolution
• Enzyme engineering
• Molecular evolution

Background:

• Genetic variation in orthologous proteins can lead to cryptic phenotypes, only apparent in changing environments.
• The molecular basis for how this variation impacts protein evolvability is not well understood.

Purpose of the Study:

• To investigate the evolutionary trajectories of four orthologous metallo-β-lactamases directed towards a new function.
• To elucidate the molecular mechanisms underlying distinct evolutionary outcomes driven by initial genetic variation.

Main Methods:

• Comparative directed evolution of four metallo-β-lactamase orthologs.
• Analysis of fitness, catalytic activity, expression, and oligomerization.
• Investigation of cryptic molecular properties and active site residue conformational variation.

Main Results:

• Different starting genotypes evolved to distinct evolutionary outcomes and fitness plateaus.
• One ortholog achieved higher fitness through increased catalytic activity, despite low initial fitness.
• Another ortholog evolved to a less-optimal outcome via changes in expression, oligomerization, and activity.

Conclusions:

• Cryptic molecular properties and conformational variation contribute to epistasis, influencing distinct evolutionary trajectories.
• Understanding the link between genetic variation and protein function is crucial for predicting protein evolution.
• This study highlights the importance of initial genotype in shaping protein evolvability.