Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase

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Abstract
Cytochrome P450 monooxygenases (P450s) are important enzymes for generating some of the enormous structural diversity of plant terpenoid secondary metabolites. In conifers, P450s are involved in the formation of a suite of diterpene resin acids (DRAs). Despite their important role in constitutive and induced oleoresin defense, a P450 gene of DRA formation has not yet been identified. By using phylogenetic cluster analysis of P450-like ESTs from loblolly pine (Pinus taeda), functional cDNA screening in yeast (Saccharomyces cerevisiae), and in vitro enzyme characterization, we cloned and identified a multifunctional and multisubstrate cytochrome P450 enzyme, CYP720B1 [abietadienol/abietadienal oxidase (PtAO)]. PtAO catalyzes an array of consecutive oxidation steps with several different diterpenol and diterpenal intermediates in loblolly pine DRA biosynthesis. Recombinant PtAO oxidized the respective carbon 18 of abietadienol, abietadienal, levopimaradienol, isopimara-7,15-dienol, isopimara-7,15-dienal, dehydroabietadienol, and dehydroabietadienal with apparent Michaelis-Menten (K(m)) values of 0.5-5.3 muM. PtAO expressed in yeast also catalyzed in vivo oxidation of abietadiene to abietic acid, but with activity much lower than with abietadienol or abietadienal. Consistent with a role of DRAs in conifer defense, PtAO transcript levels increased upon simulated insect attack using methyl jasmonate treatment of loblolly pine. The multisubstrate, multifunctional P450 diterpene oxidase PtAO, in concert with expression of a family of single-product and multiproduct diterpene synthases, allows for formation of a diverse suite of DRA defense metabolites in long-lived conifers.