Poster: Secondary metabolism
Abs #
280: Cytochrome P450 Enzyme, Abietadienol Oxidase, Catalyzes Two Oxidation Steps of Diterpene Resin Acid Biosynthesis in Loblolly Pine (Pinus taeda)
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Presenter: |
Ro, Dae-Kyun , dkro@mail.botany.ubc.ca |
Authors | Ro, Dae-Kyun (A) Arimura, Gen-Ichiro (A) Bohlmann, Joerg (A) | | Affiliations: |
(A): University of British Columbia, Department of Botany
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Abietic acid and its related isomers are the main non-volatile constituents in pine oleoresin, which serve to defend many conifer species against insect attack. The first committed step of abietic acid biosynthesis is catalyzed by a diterpene synthase to yield abietadiene, and subsequently the carbon-18 of the abietadiene hydrocarbon is oxidized via three sequential oxidations to alcohol, aldehyde and carboxylic acid. We report here the functional cloning and characterization of a novel, multifunctional cytochrome P450, abietadienol oxidase (PtAO), which catalyzes the last two oxidation steps of the in abietic acid biosynthesis in loblolly pine (Pinus taeda). The candidate P450s were selected by EST-mining aided by phylogenetic relatedness to the P450s involved in biosynthesis of Taxol or brassinosteroid. One of the candidates (PtAO) expressed in Saccharomyces cerevisiae exhibited efficient conversion of abietadinol and abietadinal, but not abietadiene, to abietic acid in in vivo feeding assays. Kinetic analyses using isolated microsomes and several diterpenoid alcohols and aldehydes as substrates substantiated the specificities of PtAO with Km values ranging from 0.5 to 5.3 μM, indicating that PtAO catalyzes oxidations of multiple substrates (i.e., double bond positional isomers of abietadienol and abietadienal). However, it could not oxidize neoabietadienol and neoabietadienal, and no compelling evidence for abietadiene hydroxylation was found. These suggest that distinct P450 genes remain to be undiscovered from pine to complete the diterpene biosynthetic pathways. In Northern-blot analysis, PtAO was induced by methyl jasmonate in bark and xylem, consistent with its defensive role against insect attack.
