Abstract
Prostaglandin H(2) (PGH(2)) is formed from arachidonic acid by cyclooxygenases (COXs) and metabolized by thromboxane (TXS) and prostacyclin synthase (PGIS), two self-sufficient cytochromes P450 (CYP). The related fungal linoleic acid (LA) biosynthetic route is catalyzed by diheme proteins of five dioxygenases (DOXs) 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 (SRSs) 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 LDSs shifted the position of intramolecular hydroxylation of 8R-hydroperoxy-LA and the heterolytic scission to towards homolytic. The self-sufficient CYP may catalyze homolytic and heterolytic cleavage of hydroperoxides and the endoperoxide of prostaglandin H(2) 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.