Structural comparisons of bifunctional fatty acid dioxygenases with allene oxide, epoxy alcohol, or diol synthase activities

Abstract

Cyclooxygenase, lipoxygenases, and other dioxygenases (DOXs) transform fatty acids to endoperoxides or hydroperoxides, which can be isomerized by cytochromes P450 (CYPs) class III. These enzymes produce biological mediators in health and disease. Prostaglandin H₂ is isomerized by thromboxane (CYP5A1) and prostacyclin synthases (CYP8A1), and hydroperoxides produced by plants by allene oxide synthases (AOSs) (Cyp74A). Bifunctional DOXs belong to the peroxidase-COX superfamily and contain CYP fusion partners. They sequentially oxidize unsaturated C₁₈ fatty acids to diols, epoxy alcohols, and allene oxides via hetero- or homolytic scissions of hydroperoxides. AlphaFold2 (AF2) models of bifunctional 8R/8S-, 9R/9S-, and 10R-DOXs illustrate the substrate recognition sites (SRSs) of the fused AOS, linoleate diol (LDS), and 10R-epoxy alcohol synthases (EAS). An Asn residue (SRS4) was positioned opposite to the heme iron of LDS, 10R-EAS, CYP8A1, and Cyp74A, but replaced at this position by Thr or nonpolar residues (Ile/Val/Ala) of 8R/9R- and 8S/9S-AOS, respectively, and by Ile of CYP5A1. Replacements of Asn and Gln residues of the SRS4 of LDSs altered the position of oxygenation and the homolytic scission of the hydroperoxide, but the amide residues were not required. The AF2 models of the CYP fusion partners illustrated the active sites in unprecedented details.

Keywords: AlphaFold2; Computed structure models; Cytochrome P450 class III; Heme peroxidase; Mutagenesis site-specific; Oxylipin biosynthesis.