Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Cai, Baochang; Siminszky, Balazs; Chappell, Joseph; Dewey, Ralph E.; Bush, Lowell P.

doi:10.1074/jbc.m112.413807

Cited by 29 publications

(19 citation statements)

References 39 publications

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“…Nicotine biosynthesis requires an oxidative deamination of N-methylputrescine, catalyzed by N-methyl putrescine oxidase (MPO1) [GenBank: AB289456.1] [17]. The conversion of nicotine to nornicotine involves N -demethylation of nicotine, catalyzed by different isotypes of a cytochrome P 450 enzyme [15], [29], [43]. Two alleles of this gene, CYP82E, were tested: Ntab CYP82E5 v 2 [GenBank: EU182719.1] is found in green leaves of N .…”

Section: Resultsmentioning

confidence: 99%

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Combination of Plant Metabolic Modules Yields Synthetic Synergies

et al. 2017

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The great potential of pharmacologically active secondary plant metabolites is often limited by low yield and availability of the producing plant. Chemical synthesis of these complex compounds is often too expensive. Plant cell fermentation offers an alternative strategy to overcome these limitations. However, production in batch cell cultures remains often inefficient. One reason might be the fact that different cell types have to interact for metabolite maturation, which is poorly mimicked in suspension cell lines. Using alkaloid metabolism of tobacco, we explore an alternative strategy, where the metabolic interactions of different cell types in a plant tissue are technically mimicked based on different plant-cell based metabolic modules. In this study, we simulate the interaction found between the nicotine secreting cells of the root and the nicotine-converting cells of the senescent leaf, generating the target compound nornicotine in the model cell line tobacco BY-2. When the nicotine demethylase NtomCYP82E4 was overexpressed in tobacco BY-2 cells, nornicotine synthesis was triggered, but only to a minor extent. However, we show here that we can improve the production of nornicotine in this cell line by feeding the precursor, nicotine. Engineering of another cell line overexpressing the key enzyme NtabMPO1 allows to stimulate accumulation and secretion of this precursor. We show that the nornicotine production of NtomCYP82E4 cells can be significantly stimulated by feeding conditioned medium from NtabMPO1 overexpressors without any negative effect on the physiology of the cells. Co-cultivation of NtomCYP82E4 with NtabMPO1 stimulated nornicotine accumulation even further, demonstrating that the physical presence of cells was superior to just feeding the conditioned medium collected from the same cells. These results provide a proof of concept that combination of different metabolic modules can improve the productivity for target compounds in plant cell fermentation.

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Section: Resultsmentioning

confidence: 99%

“…The residual NND activity in the green leaves found in the non-converter plant N . tabacum has been linked with Ntab CYP82E5 v 2 [15], [29], [43]. …”

Section: Resultsmentioning

confidence: 99%

Combination of Plant Metabolic Modules Yields Synthetic Synergies

et al. 2017

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“…Nicotine and other minor alkaloidshave been quantified in tobacco plantsusinggaschromatography [10,[13][14][15][16], electrokinetic chromatography [6,11], capillary electrophoresis (CE) [17,18], and high performance liquid chromatographic (HPLC) [5,[19][20][21][22][23][24][25][26].…”

Section: Introductionmentioning

confidence: 99%

A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

Moghbel

Ryu

Steadman

2015

Journal of Chromatography B

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“…Nornicotine, 3‐(2‐pyrrolidinyl) pyridine, is a minor alkaloid in tobacco plants. It is the N ‐demethylation product of nicotine which is a predominant tobacco alkaloid and represents 90–95% of the total alkaloid content, and this reaction occurred primarily in tobacco leaves under the mediation of nicotine demethylase enzymes . During the maturation, curing, and processing of tobacco leaves, nornicotine may be N ‐nitrosated to N ′‐nitrosonornicotine (NNN), a well characterized carcinogenic tobacco specific nitrosamines in tobacco products .…”

Section: Introductionmentioning

confidence: 99%

Enantioseparation of nornicotine in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry

Deng

Wang

Bian

et al. 2017

J of Separation Science

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Nornicotine, an alkaloid constituent of tobacco, is a precursor to the carcinogen N-nitrosonornicotine that is produced during the curing and processing of tobacco. Accumulating evidence reveals that nornicotine enantiomers have different neurochemical and behavioral effects. In the present study, an accurate and rapid method was developed for the enantioseparation of (R)-(+)-nornicotine and (S)-(-)-nornicotine enantiomers in tobacco by ultra-performance convergence chromatography with tandem mass spectrometry. Chromatographic conditions were investigated to achieve the optimal resolution of two enantiomers. Results indicated that (R)-(+)-nornicotine and (S)-(-)-nornicotine could be separated within 5 min when ammonium hydroxide was added into the cosolvent, and the best resolution (R = 4.76) was achieved on a immobilized cellulose tris-(3,5-dichlorophenylcarbamate) chiral stationary phase. The proposed method was validated and was finally applied to analyze the compositions of (R)-(+)-nornicotine and (S)-(-)-nornicotine in three typical types of tobaccos (flue-cured, burley, and oriental). It was found that, enantiomer fraction of nornicotine (the proportion of (S)-(-)-nornicotine in the nornicotine pool) in burley tobacco samples was relatively high and constant compared with flue-cured and oriental tobaccos. The effective and rapid enantioseparation of nornicotine may help the understanding of alkaloid metabolites in different tobacco varieties and may also benefit pharmacological studies of alkaloid enantiomers.

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Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Cited by 29 publications

References 39 publications

Combination of Plant Metabolic Modules Yields Synthetic Synergies

Combination of Plant Metabolic Modules Yields Synthetic Synergies

A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

Enantioseparation of nornicotine in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry

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