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. 2014 Jan 24;289(4):2013-26.
doi: 10.1074/jbc.M113.505099. Epub 2013 Dec 9.

CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy

Affiliations

CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy

Thu-Thuy T Dang et al. J Biol Chem. .

Abstract

Noscapine is a phthalideisoquinoline alkaloid investigated for its potent pharmacological properties. Although structurally elucidated more than a century ago, the biosynthesis of noscapine has not been established. Radiotracer studies have shown that noscapine is derived from the protoberberine alkaloid (S)-scoulerine and has been proposed to proceed through (S)-N-methylcanadine. However, pathway intermediates involved in the conversion of N-methylcanadine to noscapine have not been identified. We report the isolation and characterization of the cytochrome P-450 CYP82Y1, which catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine. Comparison of transcript and metabolite profiles of eight opium poppy chemotypes revealed four cytochrome P-450s, three from the CYP82 and one from the CYP719 families, that were tightly correlated with noscapine accumulation. Recombinant CYP82Y1 was the only enzyme that accepted (R,S)-N-methylcanadine as a substrate with strict specificity and high affinity. As expected, CYP82Y1 was abundantly expressed in opium poppy stems where noscapine accumulation is highest among plant organs. Suppression of CYP82Y1 using virus-induced gene silencing caused a significant reduction in the levels of noscapine, narcotoline, and a putative downstream secoberbine intermediate and also resulted in increased accumulation of the upstream pathway intermediates scoulerine, tetrahydrocolum-bamine, canadine, and N-methylcanadine. The combined biochemical and physiological data support the 1-hydroxylation of (S)-N-methylcanadine catalyzed by CYP82Y1 as the first committed step in the formation of noscapine in opium poppy.

Keywords: Biosynthesis; Cytochrome P-450; Enzyme Catalysis; Gene Silencing; Metabolism; Plant Biochemistry; Secondary Metabolism.

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Figures

FIGURE 1.
FIGURE 1.
Proposed noscapine biosynthetic pathway. Enzymes shown in blue have been functionally characterized. Dashed arrows represent several uncharacterized reactions. R1 and R2 = H or OH. Partial carbon numbering is shown for N-methylcanadine and the putative secoberbine intermediate. [O], uncharacterized oxidative conversions.
FIGURE 2.
FIGURE 2.
Heat maps showing the relative abundance of major latex alkaloids (A) and transcripts encoding selected CYPs (B) in eight opium poppy chemotypes. CYP719B1 is salutaridine synthase, CYP719A20 is stylopine synthase, and CYP80B3 is N-methylcoclaurine 3′-hydroxylase.
FIGURE 3.
FIGURE 3.
Unrooted neighbor-joining phylogenetic tree for selected CYP candidates and others shown to function in BIA metabolism. Bootstrap frequencies for each clade were based on 1,000 iterations. Abbreviations and GenBankTM accessions numbers for each protein are provided under “Experimental Procedures.”
FIGURE 4.
FIGURE 4.
Heterologous expression of CYP82X1, CYP82X2, CYP82Y1, and catalytic function of the recombinant enzymes. A, S. cerevisiae harboring pESC-leu2d::CPR (CPR), pESC-Leu2d::CYP82X1/CPR (CPR/82X1), pESC-Leu2d::CYP82X2/CPR (CPR/82X2), or pESC-leu2d::CYP82Y1/CPR (CPR/82Y1) were induced on galactose, and CPR, CYP82X1, CYP82X2, or CYP82Y1 recombinant proteins were detected using α-FLAG (CYP) and α-c-Myc (CPR) antibodies. Each lane contained 2 μg of total microsomal proteins. B, extracted ion chromatograms showing the in vivo catalytic activity of CYP82X1 (CPR/82X1), CYP82X2 (CPR/82X2), or CYP82Y1 (CPR/82Y1) using (R,S)-N-methylcanadine as the enzymatic substrate, compared with the negative control (CPR). Only the incubation of microsomal preparations containing recombinant CYP82Y1 with N-methylcanadine (m/z 354) resulted in the formation of a reaction product at m/z 370.
FIGURE 5.
FIGURE 5.
Putative identification of the CYP82Y1 reaction product as 1-hydroxy-N-methylcanadine by LTQ-Orbitrap analysis. A–D, the reaction product putatively identified as 1-hydroxy-N-methylcanadine (A) and the authentic standards narcotoline (B), N-methylcanadine (C), and hydrastine (D) each produced a single major fragment ion in MS2 at m/z 206 for 1-hydroxy-N-methylcanadine and narcotoline or m/z 190 for N-methylcanadine and hydrastine. E and F, fragmentation in MS3 of m/z 206 daughter ions of 1-hydroxy-N-methylcanadine (E) and narcotoline (F) yielded identical spectra. G and H, similarly, fragmentation of m/z 190 daughter ions of N-methylcanadine (G) and hydrastine (H) produced equivalent spectra in MS3. Arrowheads indicate parent ions.
FIGURE 6.
FIGURE 6.
Steady-state enzyme kinetics of opium poppy CYP82Y1 in total microsomal protein extracts of S. cerevisiae. Various concentrations of (R,S)-N-methylcanadine were provided as the substrate.
FIGURE 7.
FIGURE 7.
Virus-induced gene silencing confirms the involvement of CYP82Y1 in noscapine biosynthesis. A, region (gray line) of the CYP82Y1 cDNA inserted into the pTRV2 vector used to suppress CYP82Y1 transcript levels. The thick black line represents the coding region, whereas the flanking thin lines indicate noncoding 5′- and 3′-untranslated regions. Solid arrows show annealing sites of primers used to amplify cDNA fragments, whereas dotted arrows show annealing sites of primers used for qRT-PCR analysis. B, ethidium bromide-stained agarose gel showing the detection by reverse transcription-PCR of TRV coat protein transcripts in total RNA extracted from opium poppy plants infiltrated with A. tumefaciens cultures harboring pTRV1 and pTRV2 constructs. C, relative CYP82Y1 transcript abundance in control (pTRV2) and silenced (pTRV2–82Y1) plants. D, total ion chromatograms showing the major alkaloid profiles of control (pTRV2) and CYP82Y1-silenced (pTRV2–82Y1) plants. E, relative abundance of major alkaloids and other alkaloids affected by the suppression of CYP82Y1 transcript levels in control (pTRV2) and silenced (pTRV2–82Y1) plants. Asterisks represent significant differences determined using an unpaired, two-tailed Student t test (p < 0.05).
FIGURE 8.
FIGURE 8.
Effects of suppressing CYP82Y1 transcript levels by virus-induced gene silencing on the relative abundance of other putative noscapine biosynthetic genes in opium poppy. Differences in transcript abundance between control (white bars) and CYP82Y1-silenced (black bars) plants were analyzed by unpaired, two-tailed Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. 6OMT, norcoclaurine 6-O-methyltransferase; 4′OMT2, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme.
FIGURE 9.
FIGURE 9.
Relative noscapine accumulation (A) and CYP82Y1 transcript levels (B) in different opium poppy organs. cDNA synthesized using total RNA extracted from each organ was used to perform qRT-PCR. The same tissue was used for RNA and alkaloid extraction.

References

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