The crystal structure of α-Dioxygenase provides insight into diversity in the cyclooxygenase-peroxidase superfamily.

Oxylipins are potent lipid mediators generated from the oxygenation of fatty acids. Lipoxygenase (LOX) and Cyclooxygenase (COX) enzymes are predominantly responsible for the production of hydroperoxy intermediates from arachidonic acid that are further converted into eicosanoids in animals (1). Similarly, LOX enzymes generate hydroperoxy fatty acid intermediates from linoleic acid (LA; 18:2 ω-6) and α-linolenic acid (αLA; 18:3 ω-3) that are converted into jasmonates and other hormones in plants (2). Collectively, these products regulate processes involved in the maintenance, development, and response of the organism to biotic and abiotic stresses (3, 4). While LOX is found in both plant and animal kingdoms, no protein with cyclooxygenase activity has been identified in plants. In 1998, Castresana and colleagues identified a protein in tobacco leaves and Arabidopsis that was up regulated during the host defense response (5). Further characterization revealed that the protein was a member of the α-dioxygenase (α-DOX) family of heme-containing proteins that incorporate oxygen into fatty acids (6). This subfamily is part of the peroxidase-cyclooxygenase superfamily, whose members developed the ability early in the evolutionary process to generate oxidants as a general defense strategy (7). Characterization of members of this superfamily, which include COX, myeloperoxidase, eosinophil peroxidase, lactoperoxidase, thyroid peroxidase, and linoleate diol synthase (LDS), have provided insight into evolutionary relationships and allowed for critical comparisons to be made between proteins in different subfamilies. Based on its ability to oxygenate fatty acids, α-DOX is related functionally to COX and LDS, despite low sequence similarities to these enzymes. α-DOX converts LA, αLA, and other fatty acids into 2R-hydroperoxides via the stereospecific removal of the pro-R hydrogen from carbon-2 of the substrate utilizing a tyrosyl radical (Supplemental Figure S1) (8). The 2R-hydroperoxides undergo spontaneous decarboxylation to chain-shortened aldehydes, with minor amounts of 2R-hydroxy and Cn-1 fatty acids produced as well (8). In the absence of a crystal structure, COX has served as a structural surrogate for α-DOX (9, 10). Interestingly, the enzyme differs from LOX and COX by catalyzing a reaction in which oxygen addition takes place at the alpha carbon instead of at an allylic or bis-allylic position. α-DOX does not possess a strong peroxidase activity analogous to that of COX (10). We report here the 1.5A x-ray structure of A. thaliana α-DOX (Ath α-DOX). The structure represents the first member of the α-DOX family within the peroxidase-cyclooxygenase superfamily to be characterized at the molecular level. While the domain makeup of Ath α-DOX differs from COX, the active site architecture is similar between enzymes. Mutagenesis, coupled with functional analyses, reveals that His-318, Thr-323, and Arg-566 are critical determinants for oxygenase activity. Defining the location of these residues within the active site has allowed us to propose a model for productive substrate binding that provides insight into the ability of Ath α-DOX to oxygenate a variety of fatty acids. Finally, identification of two, large inserts located near the heme explain the reduced peroxidase enzyme observed for the enzyme.