Structural Comparisons of Bifunctional Fatty Acid Dioxygenases with Allene Oxide, Epoxy Alcohol, or Diol Synthase Activities

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Fatty acids are converted into biologically active mediators by enzymes such as cyclooxygenases, lipoxygenases, and dioxygenases (DOXs).
• Cytochromes P450 (CYPs) class III enzymes further isomerize these fatty acid derivatives, playing crucial roles in both health and disease.
• Specific examples include thromboxane (CYP5A1) and prostacyclin synthases (CYP8A1) in prostaglandin isomerization and allene oxide synthases (AOS) (Cyp74A) in plant hydroperoxide metabolism.

Purpose of the Study:

• To investigate the structural basis of substrate recognition and catalytic mechanisms in bifunctional dioxygenases (DOXs) and their cytochrome P450 (CYP) fusion partners.
• To utilize AlphaFold2 (AF2) modeling to visualize the active sites and substrate recognition sites (SRSs) of various DOXs and CYPs involved in fatty acid metabolism.

Main Methods:

• Generation of AlphaFold2 (AF2) models for bifunctional 8R/8S-, 9R/9S-, and 10R-DOXs, as well as their fused CYP partners.
• Analysis of the substrate recognition sites (SRSs), particularly SRS4, focusing on key amino acid residues (Asn, Thr, Ile, Val, Ala) positioned near the heme iron.
• Investigating the impact of specific residue substitutions (e.g., Asn, Gln) in SRS4 on oxygenation patterns and hydroperoxide scission.

Main Results:

• AF2 models provided detailed insights into the active sites of fused AOS, linoleate diol synthases (LDS), and 10R-epoxy alcohol synthases (EAS).
• Identified a conserved Asn residue at SRS4 opposite the heme iron in LDS, 10R-EAS, CYP8A1, and Cyp74A, with variations (Thr, Ile, Val, Ala) in other AOS and CYP5A1.
• Demonstrated that replacements of Asn and Gln residues in LDS SRS4 alter oxygenation and scission, but these amide residues are not essential for catalysis.

Conclusions:

• AlphaFold2 models offer unprecedented structural detail of the active sites in CYP fusion partners of bifunctional DOXs.
• The identified amino acid variations in SRS4 are critical for determining substrate specificity and reaction outcomes in these enzymes.
• Understanding these structural-functional relationships advances knowledge of fatty acid metabolism and the production of vital biological mediators.