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J Biol Chem, Vol. 274, Issue 47, 33764-33770, November 19, 1999
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 20.3
6 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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