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

Abstract

conifer defense ͉ gibberellic acid ͉ diterpene resin acids ͉ conifer genomics ͉ plant secondary metabolism D iterpene resin acids (DRAs) (Fig. 1A) are important defense compounds of conifers against potential herbivores and pathogens, such as bark beetles and their associated fungi (1-3). DRAs are formed and sequestered as major components of complex oleoresin blends in resin ducts, resin blisters, or resin cells in stems, needles, and roots of most conifers. Biosynthesis of DRAs involves formation of geranylgeranyl diphosphate (GGDP), cyclization of GGDP to a series of diterpene olefins by activity of diterpene synthases (diTPSs), and three subsequent oxidations at carbon 18 (Fig. 1B). A small family of singleproduct or multiproduct diTPSs of DRA biosynthesis has recently been cloned and characterized (4-6). Studies with grand fir (Abies grandis) and lodgepole pine (Pinus contorta) tissue extracts showed that stepwise oxidation of the diterpene olefin abietadiene (1a) to abietic acid (1d) can be achieved by membrane-bound cytochrome P450 monooxygenase (P450) and soluble aldehyde dehydrogenase enzyme activities (Fig. 1B) (7, 8). The general pathway scheme of oxidation of abietadiene to abietic acid in conifer secondary metabolism resembles that of oxidation of ent-kaurene to ent-kaurenoic acid in the biosynthesis of gibberellin phytohormones (9-11). A multifunctional P450 responsible for the three-step oxidation of ent-kaurene to kaurenoic acid has previously been identified by using genetic approaches in Arabidopsis thaliana (10-11). Similarly, a multifunctional P450 also catalyzes the subsequent three-step oxidation from kaurenoic acid to GA 12 in Arabidopsis (12). Cloning and identification of P450s of DRA secondary metabolism have been hampered by difficulties in purifying the corresponding enzymes and by lack of suitable forward genetic tools for conifers. However, large collections of ESTs for loblolly pine (Pinus taeda, Pt) (13) can enable gene discovery and biochemical identification of candidate P450 cDNAs of terpenoid secondary metabolism in conifers. Here, we describe cloning and functional characterization of abietadienol͞abietadienal oxidase (PtAO, CYP720B1), a P450 enzyme that is unusual in that it catalyzes an array of consecutive oxidations of multiple diterpene alcohol and aldehyde intermediates in DRA biosynthesis in loblolly pine. The methyl jasmonate (MJ)-inducible PtAO can account for much of the oxidative diversification of diterpenoid natural product defense compounds in loblolly pine. Materials and MethodsMaterials. Seedlings of loblolly pine and Sitka spruce (Picea sitchensis) were grown to 2-year-old trees as described in ref. 14. Yeast strain YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-⌬63, his3-⌬200, and leu2-⌬1) and yeast dual expression vectors (pESC-Leu and pESC-His) were from Stratagene. Diterpenoid substrates were prepared from the corresponding DRAs (Helix Biotech, Surrey, BC, Canada) as described in Supporting Materials and Methods, which is published as supporting informat...

The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol

“…In prior research, the in vivo production of diterpene resin acids in metabolically engineered yeast containing the genes encoding geranylgeranyl diphosphate synthase and PaLAS produced only abietadiene and none of the other diterpenes previously found in the multi-product profiles of LAS-type enzymes (3). At the time, this simpler product profile was rationalized as the lower pH of the yeast culture medium affecting the enzyme product profile.…”

“…This step may occur prior to the oxidation at carbon-18 by cytochromes P450 of the CYP720B subfamily such as CYP720B1 (3,8) or after.…”

“…Abietadiene and abietadienol were prepared by Best West Laboratories Inc. (Salt Lake City, UT) from abietic acid as described previously (3). The synthesis of (Ϯ)-13-hydroxy-8(14)-abietene from (Ϯ)-␣-cyclocitral (Toronto Research Chemicals, North York, Ontario, Canada) was attempted by Best West Laboratories Inc. as described previously (19) with the exception of the final isopropylation step, for which the method of Gu et al (20) was used.…”

“…To date, only two types of diterpene synthases have been characterized in conifer resin acid biosynthesis: those that produce isopimaradiene and sandaracopimaradiene (4,5) and those that produce a mixture of abietadiene, levopimaradiene, neoabietadiene, and palustradiene (4 -7). So far, only one cytochrome P450 of diterpene resin acid biosynthesis, CYP720B1 from loblolly pine (Pinus taeda), has been reported to oxidize a small subset of these diterpenes (3). These three enzymes are not sufficient to generate the structural and functional diversity of the diterpene resin acids observed in conifer resin (2), requiring the further characterization of these and other enzymes (4,8).…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…In contrast to this divergent evolution, the emergence of a large family in the CYP85 clan, CYP720B, encoding P450s catalysing the three-step oxidation of diterpene olefins, indicates convergent evolution of this function in the CYP85 and CYP71 clan [47,51]. Additionally, members of this family have acquired a certain degree of promiscuity, accepting with varying affinity a range of structurally related diterpene olefins, alcohols and aldehydes, which is in stark contrast to the known activities of the enzymes of general phytohormone metabolism (figure 4) [47,52]. While the plasticity of individual P450s of the CYP720B family contributes to chemical diversity in the defence-related diterpene resin acids, functional characterization of divergent members of the family encompassing at least a dozen P450s will shed light on the evolutionary advantage of the observed genetic diversity.…”