Nicotine demethylation in Nicotiana cell suspension cultures: N′-formylnornicotine is not involved

Bartholomeusz, Trixie Ann; Bhogal, Ramneek K; Molinié, Roland; Felpin, François‐Xavier; Mathé-Allainmat, Monique; Meier, Anna-Carolin; Dräger, Birgit; Lebreton, Jacques; Roscher, Albrecht; Robins, Richard J.; Mesnard, François

doi:10.1016/j.phytochem.2005.07.005

Cited by 23 publications

(20 citation statements)

References 21 publications

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“…Previous isotope-labeling experiments have shown that the methyl group derived from nicotine is recycled back into the folate pool (Mesnard et al, 2002;Bartholomeusz et al, 2005). These studies, together with the altered alkaloid profiles in the transgenic lines described here, lead us to speculate that C1 units may mediate the regulation of NtMTHFR1 on CYP82E4 (Fig.…”

Section: Discussionsupporting

confidence: 57%

“…On the other hand, it has been reported that the above three alkaloids can undergo enzymatic demethylation (Dawson, 1945;Skurský et al, 1969;Miller et al, 1973). Isotopelabeling studies in Nicotiana plumbaginifolia suspension cells have shown that methyl groups released from nicotine by N-demethylation find their way into many primary metabolites, such as Met and Ser, suggesting that they reenter the C1-folate pool (Mesnard et al, 2002;Bartholomeusz et al, 2005). Although it is well documented that C1 units from the THF-mediated pathway are required for alkaloid biosynthesis in plants (Hanson et al, 2000), it is unknown whether any molecular mechanisms are involved in regulating alkaloid N-demethylation in response to the C1 demand.…”

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confidence: 99%

“…Nornicotine is the second most abundant alkaloid in most tobacco cultivars (Saitoh et al, 1985), and its conversion is mainly controlled by the nicotine N-demethylase gene CYP82E4 (Siminszky et al, 2005;Xu et al, 2007). It is believed that CYP82E4-mediated oxidative N-demethylation of nicotine produces an undefined unstable intermediate, which then breaks down spontaneously to nornicotine and formaldehyde (Mesnard et al, 2002;Bartholomeusz et al, 2005;Siminszky et al, 2005;Molinié et al, 2007). Mesnard et al (2002) suggested that the formaldehyde released from nicotine is recycled back into the THFmediated C1 pool.…”

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confidence: 99%

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Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

et al. 2012

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Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-tonornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and Sadenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine Ndemethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions. Tetrahydrofolate (THF)-mediated one-carbon (C1) metabolism generates and provides C1 units in different oxidation states for various anabolic pathways, including alkaloid biosynthesis in plants (Hanson et al., 2000). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylene-THF into the most reduced C1 derivative, 5-methyl-THF (Guenther et al., 1999;Roje et al., 1999;Gelling et al., 2004). The latter then serves as the methyl group donor for Met synthesis from homocysteine (Guenther et al., 1999;Hanson et al., 2000). More than 80% of synthesized Met is further converted to S-adenosyl-methionine (AdoMet), which is the universal methyl group donor in reactions leading to modifications of various primary and secondary metabolites (Giovanelli et al., 1985;Hanson et al., 2000;Roje, 2006). In plants, MTHFRs have been cloned and characterized from Arabidopsis (Arabidopsis thaliana) and maize (Zea mays; Roje et al., 1999). In contrast to the mammalian enzymes, which are NADPH dependent and inhibited by AdoMet, plant MTHFRs are NADH dependent and AdoMet insensitive. In addition, the reaction that they catalyze is likely reversible in the cytosol (Roje et al., 1999). To 1 This work was supported by the Golden LEAF Foundation (a startup fund to the Biomanufacturing Research Institute & Technology Enterprise), the National Institute of General Medical Sciences (grant no. SC3GM088084 to J.X.), the National Science Foundation of China and the Yunnan Tobacco Company (grant nos. 31060046 and 2011YN04 to B.X.), and the National Science Foundation (grant nos. MCB-04...

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Section: Discussionsupporting

confidence: 57%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

et al. 2012

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“…Therefore, LolP1 is apparently a cytochrome P450 monooxygenase that probably catalyzes oxidation of an N-methyl group in NML to the N-formyl group in NFL. LolP-catalyzed oxygenation of NML rules out alternative reaction mechanisms for formation of NFL, such as condensation of formaldehyde with the C-1 amine in loline, a reaction suggested for the formation of N-formylnornicotine, from nornicotine (Bartholomeusz et al, 2005). At least two oxygenations are required for the NML-to-NFL conversion: hydroxylation of the N-methyl group giving an N-methylhydroxyl group followed by dehydrogenation of the alcohol to give the N-formyl group (Fig.…”

Section: Discussionmentioning

confidence: 99%

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

Spiering

Faulkner

Zhang

et al. 2008

Fungal Genetics and Biology

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“…13 In order to further probe nicotine degradative enzymatic reaction mechanisms by use of the associated KIE effects, we have prepared nicotine doubly labelled on the pyridine ring and the N-methyl of the pyrrolidine substituent. Such doubly labelled species provide an internal reference peak from the 2 H aromatic signal, allowing better precision in determining the ( …”

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confidence: 99%

Synthesis of four racemic nicotine isotopomers doubly labelled with stable isotope

Hatton

Silvestre

Robins

et al. 2009

Labelled Comp Radiopharmac

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Four racemic nicotine isotopomers, doubly labelled with stable isotope, were prepared for use in studies of plant metabolism. First, starting from halogeno nicotinates, one deuterium was introduced into the pyridine moiety by reductive dehalogenation with zinc, and choosing deuterated acetic acid as the acidic medium. Pyridine 2 H-labelled nornicotine derivatives were then formed in two reaction steps, a condensation reaction with N-vinylpyrrolidinone followed by catalytic hydrogenation. The deuteromethylation of these monodeuterated nornicotines was achieved by reduction of the corresponding ethyl carbamates with deuterated lithium aluminium hydride. The four regioisotopomers obtained were at the least 90% labelled on each site, sufficient for the metabolic studies envisaged.

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Nicotine demethylation in Nicotiana cell suspension cultures: N′-formylnornicotine is not involved

Cited by 23 publications

References 21 publications

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

Synthesis of four racemic nicotine isotopomers doubly labelled with stable isotope

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