Identification of a Jasmonate-regulated Allene Oxide Synthase That Metabolizes 9-Hydroperoxides of Linoleic and Linolenic Acids

Abstract

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9-and 13-hydroperoxides of linoleic and linolenic acid to ␣-ketol, ␥-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was Ϸ10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.Oxylipins comprise a group of biologically active compounds that are produced by oxidative metabolism of polyunsaturated fatty acids. Members of the eicosanoid family of lipid mediators have been studied extensively with respect to their biosynthesis from arachidonic acid and their function in diverse physiological processes in animal cells (1). In plants, which lack arachidonic acid, oxygenated derivatives of C 18 fatty acids participate in the regulation of many defense-related and developmental processes. The biosynthesis of most phytooxylipins is initiated by lipoxygenase (LOX), 1 which adds molecular oxygen to either the C-9 or C-13 position of linolenic or linoleic acid (2). The resulting hydroperoxides are further metabolized by several enzymes including three closely related members of the CYP74 family of cytochromes P-450: allene oxide synthase (AOS), hydroperoxide lyase (HPL), and divinyl ether synthase (DES). Indeed, much of the structural and functional diversity in oxylipin metabolism in plants can be accounted for by the activity of CYP74s that metabolize 9-and 13-hydroperoxides to a wide range of products (3). In contrast to typical P-450 monooxygenases, CYP74 P-450s do not require O 2 and a NADPHdependent P-450 reductase for activity. Rather, they use a hydroperoxide group both as the oxygen donor and as a source of reducing equivalents (4, 5). This unique catalytic feature is shared by thromboxane synthase and prostacyclin synthase, two P-450 enzymes involved in the synthesis of eicosanoids (6). A greater understanding of the biochemical and physiological function of this atypical class of P-450 enzymes promises to provide new insight into the evolution ...

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…The analysis of the DNA sequences revealed open reading frames which encode a 530 (AOS1), a 509 (AOS2), and a 491 amino acid protein (AOS3) with calculated molecular weights of 59.6, 57.3, and 55.7 kDa, respectively. The amino acid sequences showed the highest similarity (over 90 % amino acid identity) with the published LeAOS1 , LeAOS2 , and LeAOS3 (Itoh et al, 2002). Because of this homology and the biochemical characteristics (see below), the enzymes were named StAOS1-3 (Solanum tuberosum AOS1-3) and classified in the CYP74A (StAOS1 and 2) or CYP74C subfamily (StAOS3), respectively.…”

“…Among the CYP74 family enzymes, only a few prefer 9-hydroperoxides, including DES enzymes from tomato and potato which produce colneleic and colnelenic acid ) and the recently cloned AOS3 from tomato (LeAOS3) (Itoh et al, 2002). Concerning the endogenous amounts of 9-CYP74 products in plants, information about aldehydes ((3Z)-nonenal, (3Z,6Z)-nonadienal) and divinylethers (colneleic and colnelenic acid) is available (Weber et al, 1999;Göbel et al, 2002;Göbel et al, 2003), whereas 9-AOS products have not been analyzed to date.…”

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site‐directed mutagenesis

“…The ratio of 9-oxononanoic acid to a-ketol produced by S297A mutant was increased about 10 3 -fold comparing to the WT LeAOS3. The WT LeAOS3 utilizes both 9-HPOD and 13-HPOD [14,15]. In contrast, both mutant forms F295I and S297A utilized preferentially 9-HPOD.…”

“…The His-tagged recombinant LeAOS3 (WT and mutant forms) were expressed and prepared as described before [14]. His-tagged recombinant proteins were purified by immobilized metal affinity chromatography (IMAC) using the cobalt TALON metal affinity resin (Clontech) as described before [15].…”

“…Abbreviations: AOS, allene oxide synthase; HPL, hydroperoxide lyase; DES, divinyl ether synthase; WT, wild type; IHCD, I-helix central domain; SRS, substrate recognition site; 9-HPOD, (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid; 13-HPOD, (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid; IMAC, immobilized metal affinity chromatography; TMS, trimethylsilyl concentration was estimated as described [14]. The enzyme activity was measured with Varian Cary 50 spectrophotometer by the decrease of 9-HPOD absorbance at 234 nm [15].…”