Catalytic properties of allene oxide synthase from flaxseed (Linum usitatissimum L.)

Abstract

We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 4.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)- and 13(S)-hydroperoxides of linoleic and linolenic acid were determined by a photometric assay. 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9 +/- 5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 microM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable alpha-ketol hydrolysis products.

On the specificity of allene oxide cyclase

“…The substrate specificities shown here for the corn enzyme are similar to those of the recombinant AOS from barley leaves (Maucher, H., Hause, B., Feussner, I., Ziegler, J., and Wasternack, C., in preparation) and guayule (31), where a steady decrease in K m and v max in the order 13(S)-HPOD > 13(S)-HPOT > 15(S)-HPETE was also observed. The AOS from flax showed only minor differences in affinity toward 13(S)-HPOD, 9(S)-HPOD, and 9(S)-HPOT but, in contrast to AOS from corn and barley, converted the 13-hydroperoxide at a rate almost 40-fold higher than that of the 9-hydroperoxides (32).…”

Rice HYDROPEROXIDE LYASES with Unique Expression Patterns Generate Distinct Aldehyde Signatures in Arabidopsis

“…The AOS enzymes within the CYP74A subfamily are also classified, based on their substrate preference, into 13-and 9-/13-AOSs (Laudert et al, 1996;Schneider and Schreier, 1998;Howe et al, 2000;Maucher et al, 2000;Sivasankar et al, 2000;Feussner and Wasternack, 2002;Maucher et al, 2004). To explore the possible presence of a common distinctive signature residue(s) involved in the substrate specificity and/or preference of HPLs and AOSs, we further performed multiple sequence alignments of the catalytic sites of all characterized members of these enzymes.…”

Identification of a Component that Induces Flowering of Lemna among the Reaction Products of α-Ketol Linolenic Acid (FIF) and Norepinephrine

“…The 9-hydroxyl group was found to be predominantly 9R, with an enantiomeric excess of 40% (70% 9R and 30% 9S). It has been reported that allene oxide synthase prepared from flax seed transformed the 13-hydroperoxide of linoleic acid and linolenic acid into a-ketol with an (R) to (S) configuration ratio of 7 : 3 (Schneider and Schreier 1998). Also, in the case of the 9-hydroperoxide of linolenic acid, allene oxide synthase generated a mixture of R-and S-FIF.…”