Ancestral Sequence Reconstruction of the Ethylene-Forming Enzyme

Area of Science:

• Biochemistry
• Enzymology
• Evolutionary Biology

Background:

• The ethylene-forming enzyme (EFE) exhibits diverse catalytic activities, including 2-oxoglutarate (2OG) conversion to ethylene and 2OG oxidative decarboxylation coupled with l-arginine (l-Arg) hydroxylation.
• Understanding the evolutionary trajectory of EFE's multiple functions is crucial for deciphering enzyme adaptation.

Purpose of the Study:

• To investigate the evolutionary history of the ethylene-forming enzyme (EFE) and its diverse catalytic activities.
• To elucidate the structural and functional basis for EFE's distinct reactions, including ethylene formation and l-arginine hydroxylation.

Main Methods:

• Reconstruction and analysis of 11 ancestral EFE proteins.
• X-ray crystallography and AlphaFold2 modeling for structural determination of ancestral proteins.
• Biochemical assays to assess the reactivity of extant and ancestral EFEs.

Main Results:

• Two extant enzymes and reconstructed ancestors were shown to produce ethylene and 3-hydroxypropionate (3HP).
• Structural analysis revealed critical residues in the 2OG and l-Arg binding pockets influencing enzyme activity.
• Ancestral analysis indicated the primordial EFE possessed both ethylene-forming and l-Arg hydroxylation activities concurrently.

Conclusions:

• The ethylene-forming enzyme's ancestral form likely possessed both ethylene-forming and l-arginine hydroxylation activities.
• Enzyme evolution involved modifications in substrate-binding pockets, particularly for 2OG and l-Arg interactions.
• The study provides insights into the evolutionary pathways of multifunctional enzymes.