Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla.
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 micromol/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 Ca(2+). The resulting rates of reaction are approximately 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 approximately 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 20.3omega6 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 H(2)O(2); catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE.
Boutaud
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Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602, USA.
Brash
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AR
D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003063:Cnidaria / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D008084:Lipoxygenase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012492:Salts; D013379:Substrate Specificity
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D012492:Salts; D002374:Catalase; D008084:Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase