Abstract
Catalase-related allene oxide synthase (cAOS) is a hemoprotein that converts a specific fatty acid hydroperoxide to an unstable allene oxide intermediate at turnover rates in the order of 1000 per second. Fatty acid allene oxides are intermediates in the formation of cyclopentenone or hydrolytic products in marine systems, most notably the prostanoid-related clavulones. Although the key catalytic amino acid residues around the active site of cAOS are the same as in true catalases, cAOS does not react with hydrogen peroxide. cAOS occurs exclusively as the N-terminal domain of a naturally occurring fusion protein with a C-terminal lipoxygenase (LOX) domain that supplies the hydroperoxide substrate. In marine invertebrates, an 8R-LOX domain converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) and the cAOS domain forms an 8,9-epoxy allene oxide. The fusion protein from the sea whip octocoral Plexaura homomalla is the prototypical model with crystal structures of the individual domains. The cAOS (43kDa) expresses exceptionally well in Escherichia coli, with yields of up to 100mg/L. This article describes in detail expression and assay of the P. homomalla cAOS and two methods for the preparation of its 8R-HPETE substrate. Another article in this volume focuses on the P. homomalla 8R-LOX (Gilbert, Neau, & Newcomer, 2018).