What question did this study set out to answer?

The research aims to compare the catalytic and structural characteristics of linoleate dioxygenases and cytochrome P450 enzymes within the prostaglandin synthesis pathway.

February 11, 2026Open Access

Catalytic and Structural Comparisons of Linoleate Dioxygenases and their Cytochrome P450 Companions with Enzymes of the Cyclooxygenase Cascade

Puntos clave

The research aims to compare the catalytic and structural characteristics of linoleate dioxygenases and cytochrome P450 enzymes within the prostaglandin synthesis pathway.
Utilized AlphaFold2 for predicting 3D structures of dioxygenase-cytochrome P450 domains.
Conducted superposition analyses of enzyme complex structures.
Analyzed amino acid residues in substrate recognition sites and catalytic mechanisms.
Structural similarities in alpha-helices and recognition sites were found between dioxygenases and cyclooxygenases.
A specific Asn residue in the substrate recognition site of certain dioxygenases was noted for its catalytic implications.
Heterolytic vs. homolytic cleavage mechanisms are influenced by metal centers in the enzymes.

Resumen

Prostaglandin H2 (PGH2) is formed from arachidonic acid by cyclooxygenases (COX) and metabolized by thromboxane (TXS) and prostacyclin synthases (PGIS), two self-sufficient cytochromes P450 (CYP). The related fungal linoleic acid (LA) biosynthetic route is catalyzed by di-heme proteins of five dioxygenases (DOX) fused to three akin CYP subfamilies of allene oxide (AOS), linoleate diol (LDS), and epoxy alcohol (EAS) synthases. AlphaFold2 predicted the 3D structures of the DOX-CYP domains with very high confidence. Superposition with the COX:LA enzyme complex indicated that the protein fold of central α-helices and the motifs of the substrate recognition sites (SRS) were conserved, which suggest evolution from an ancient peroxidase precursor. TXS, PGIS, and AOS catalyze homolytic scissions of oxygen-oxygen bonds and LDS/EAS heterolytic scissions. The SRS4 of LDS and EAS predicted an Asn residue at close distal axial position of the heme thiolate iron in analogy with PGIS and plant AOS, but a nonpolar in TXS and 8S/9S-AOS, and a polar (Thr) in 8R/9R-AOS. Replacements of amide residues in SRS4 of LDS shifted the position of intramolecular hydroxylation of 8R-hydroperoxy-LA and the heterolytic scission to towards homolytic. The self-sufficient CYP may catalyze homo- and heterolytic cleavage of hydroperoxides and the endoperoxide of PGH2 by different mechanisms, but the presentation of the oxygen-oxygen bonds to the metal centers might be crucial. The AF2 models illustrate the structural, catalytical, and evolutionary relationships between COX and microbiological DOX with CYP companions in unprecedented details, which reveal multiple amino acids of potential catalytic significance for future research.

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