Intricate Evolution of Multifunctional Lipoxygenase in Red Algae

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Lower organism lipoxygenases (LOXs) exhibit diverse substrate and functional versatility in fatty acid oxidation.
• The evolutionary mechanisms enabling multifunctional catalytic domains in LOXs remain unclear.

Purpose of the Study:

• To investigate the structural and evolutionary basis of multifunctional lipoxygenase (LOX) activity in the red alga *Pyropia haitanensis* (PhLOX).
• To elucidate how PhLOX integrates hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activities within a single catalytic domain.

Main Methods:

• Molecular docking and site-directed mutagenesis were employed to identify key amino acid residues.
• Phylogenetic analysis was conducted to trace evolutionary relationships.
• Functional analysis of homologous LOXs from *Shewanella violacea* and *Chondrus crispus* was performed.

Main Results:

• Specific residues (Phe642, Phe826) regulate fatty acid peroxidation, while others (Gln777, Asn575) are crucial for AOS function.
• Mutations to leucine at Asn575, Gln777, or Phe826 enhanced HPL activity.
• Phylogenetic analysis revealed unique sites (Asn575, Phe826) in PhLOX clades and conserved residues (Phe642, Gln777) in other LOXs.
• The N-terminal SRPBCC domain was essential for PhLOX's functional versatility.
• HPL activity appears ancestral, while AOS function was acquired via active pocket mutations.

Conclusions:

• PhLOX achieved its dual HPL and AOS activities through a combination of ancestral functional domains and unique amino acid substitutions.
• Red algal LOXs evolved versatility by integrating existing domains and acquiring specific mutations within the active site.