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J Biol Chem, Vol. 274, Issue 47, 33764-33770, November 19, 1999

Purification and Catalytic Activities of the Two Domains of the Allene Oxide Synthase-Lipoxygenase Fusion Protein of the Coral Plexaura homomalla

From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602

The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 µmol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mM Na⁺, Li⁺, or K⁺), and an additional 5-fold by 10 mM Ca²⁺. The resulting rates of reaction are 300 turnovers/s, 1-2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of 1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.36 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H₂O₂; catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE.

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