Enzyme Evolution: An Epistatic Ratchet versus a Smooth Reversible Transition

Area of Science:

• Biochemistry
• Evolutionary Biology
• Structural Biology

Background:

• Evolutionary trajectories are generally irreversible, with reverted mutations often leading to loss of ancestral and novel functions.
• Understanding the structural and mechanistic basis for reversion compatibility versus incompatibility is crucial for evolutionary studies.

Purpose of the Study:

• To investigate the causes of reversion compatibility and incompatibility in protein evolution using mammalian paraoxonase-1 (PON1).
• To compare two distinct evolutionary trajectories: neo-functionalization towards organophosphate hydrolase (OPH) activity and re-functionalization towards native lactonase activity.

Main Methods:

• Laboratory evolution of mammalian paraoxonase-1 (PON1).
• Analysis of His115 revertants from neo-functionalization and re-functionalization trajectories.
• Determination of crystal structures and molecular simulations of protein variants.

Main Results:

• Revertants from the neo-functionalization trajectory lost both OPH and ancestral lactonase activities.
• Revertants from the re-functionalization trajectory fully restored lactonase activity.
• Structural analysis revealed displaced catalytic residues in the OPH-evolved variant, causing loss of both functions, while mechanistic versatility in the lactonase-restored variant allowed for compatible reversions.

Conclusions:

• Reversion compatibility in protein evolution depends on the specific trajectory and underlying mechanistic flexibility.
• Mechanistic versatility allows for sequence-reversible compositions, enabling restoration of ancestral functions even after significant evolutionary divergence.
• Repurposing active sites for new functions can lead to evolutionary pathways with incompatible reversions.