Pervasive cryptic epistasis in molecular evolution.

Area of Science:

• Biochemistry and Molecular Evolution
• Enzyme Function and Adaptation

Background:

• The functional consequences of most amino acid substitutions during molecular evolution remain largely unknown due to the vast number of possibilities.
• Investigating enzyme evolution requires understanding how mutations affect protein function and how these effects are compensated.

Purpose of the Study:

• To investigate the functional impact of naturally occurring amino acid differences between Escherichia coli and Pseudomonas aeruginosa isopropymalate dehydrogenase (IMDH).
• To explore the role of epistasis in enzyme adaptation and molecular evolution.

Main Methods:

• Generated 168 single amino acid mutations in E. coli IMDH, mimicking differences found in P. aeruginosa IMDH.
• Assessed the functional activity of mutant IMDH enzymes.
• Screened for compensatory mutations at specific sites near the active site and in a different domain.

Main Results:

• A majority of mutations (104/168) had neutral effects, while 63 compromised function and one enhanced it.
• Transition to P. aeruginosa IMDH function requires extensive epistasis to overcome deleterious mutations, contradicting independent site evolution.
• Compensatory mutations were found at a site distant from the active site, demonstrating long-range epistatic interactions (>20Å).

Conclusions:

• Molecular evolution often involves complex epistatic interactions, where mutations at one site can compensate for effects at distant sites.
• The independent site evolution assumption in molecular phylogenetics is challenged by findings in IMDH evolution.
• Epistasis plays a critical role in enabling the fixation of mutations that would otherwise be detrimental, facilitating adaptation.