Diversity and Evolution of Plant P450 and P450-Reductases

Durst, Francis; Nelson, David R.

doi:10.1515/dmdi.1995.12.3-4.189

Cited by 161 publications

(118 citation statements)

References 18 publications

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“…29 and 30 and references therein). Phylogenetic analysis placed CYP720B1͞B2 and CYP716B1͞B2 into the CYP85 clan (31) containing several known P450s of diterpenoid (taxol and gibberellin) and triterpernoid (brassinosteroid) metabolism (see Fig. 7, which is published as supporting information on the PNAS web site).…”

Section: Resultsmentioning

confidence: 99%

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

Arimura

Lau

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

171

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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...

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Section: Resultsmentioning

confidence: 99%

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

Arimura

Lau

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

171

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“…A phylogenetic analysis of all characterized CYP79 enzymes and the four P. trichocarpa CYP79 sequences showed that the poplar CYP79 proteins group together with other members of the CYP79D subfamily (see Supplemental Figure 2 online), which were described to be involved in the biosynthesis of cyanogenic glycosides (Andersen et al, 2000;Forslund et al, 2004). Motifs reported to be conserved in nearly all P450 enzymes, such as the ProProxxPro motif right before the N-terminal membrane anchor, the heme binding site including the conserved Cys residue, and the ProGluArgPhe motif (Durst and Nelson, 1995), were also found in the P. trichocarpa sequences (see Supplemental Figure 3 online). Moreover, the heme binding site as well as the ProGluArgPhe motif showed substitutions unique for the CYP79 family.…”

Section: L-phe Is the Precursor Of Aromatic Nitrogenous Poplar Volatilesmentioning

confidence: 99%

“…Moreover, the heme binding site as well as the ProGluArgPhe motif showed substitutions unique for the CYP79 family. In comparison to the consensus sequence ProPheGlyxGlyArgArgxCysxGly described for A-type P450s (Durst and Nelson, 1995), the heme binding motifs of both CYP79D6v3 and CYP79D7v2 (457-SerPheSerThrGlyLysArgGlyCysIleGly-467; see Supplemental Figure 3 online) contained three amino acid substitutions which have been reported for other members of the CYP79 family (Bak et al, 1998a;Andersen et al, 2000;Nielsen and Møller, 2000). The ProGluArgPhe motif (residues 434 to 440; see Supplemental Figure 3 online) was altered to ProGluArgHis with a His substitution being unique for the CYP79 family (Bak et al, 1998a).…”

Section: L-phe Is the Precursor Of Aromatic Nitrogenous Poplar Volatilesmentioning

confidence: 99%

“…The ProGluArgPhe motif (residues 434 to 440; see Supplemental Figure 3 online) was altered to ProGluArgHis with a His substitution being unique for the CYP79 family (Bak et al, 1998a). The replacement of two conserved Thr-(Thr/Ser) residues in a proposed substrate binding pocket (Durst and Nelson, 1995) with Asn-Pro (see Supplemental Figure 3 online; residues 328/ 329 for CYP79D6) is also a typical feature for enzymes of the CYP79 family.…”

Section: L-phe Is the Precursor Of Aromatic Nitrogenous Poplar Volatilesmentioning

confidence: 99%

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Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

et al. 2013

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Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus 3 canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.

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“…The Arabidopsis genome contains 272 cytochrome P450 genes (including 26 pseudogenes; Rhee et al, 2003;Wortman et al, 2003) of which 33 have been grouped together in the CYP86 clan of non-A-type P450s, which include the subfamilies CYP86, CYP94, CYP96, and CYP704 (Durst and Nelson, 1995;Nelson et al, 2004). Members from this clan have previously been shown to be involved in the hydroxylation reactions of fatty acyl-derived substrates (Kahn et al, 2001;Wellesen et al, 2001;Werck-Reichhart et al, 2002) and therefore genes within this clan may be regarded as candidates for cuticle biosynthetic enzymes.…”

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confidence: 99%

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

et al. 2007

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Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.

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Diversity and Evolution of Plant P450 and P450-Reductases

Cited by 161 publications

References 18 publications

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

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

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

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