Evidence for the involvement of tetrahydrofolate in the demethylation of nicotine by Nicotiana plumbaginifolia cell-suspension cultures

Abstract

The conversion of nicotine to nornicotine by Nicotiana plumbaginifolia Viv. cells was investigated by analysing the redistribution of label during feeding experiments with (R,S)-[2H- methyl]nicotine, (R,S)-[13C- methyl]nicotine and (R,S)-[14C- methyl]nicotine, and the results show that the N-methyl group of nicotine can be recycled into primary metabolism. Nuclear magnetic resonance (NMR) analysis of ethanolic extracts of cells grown in the presence of (R,S)-[13C- methyl]nicotine, using 1H-13C correlation spectroscopy (HMQC, HMBC), revealed the presence of [3-13C]serine and [13C- methyl]methionine. Label was also identified in a cysteinyl derivative and in several methoxylated compounds, but no evidence was obtained with either NMR or ion-trap mass spectrometry for the presence of any intermediate between nicotine and nornicotine. However, experiments with (R,S)-[14C- methyl]nicotine indicated that 70-75% of the metabolised label was released as carbon dioxide. These results are consistent with a pathway in which the oxidative hydrolysis of the nicotine methyl produces an unstable intermediate, N'-hydroxymethylnornicotine, that breaks down spontaneously to nornicotine and formaldehyde, with the formaldehyde being metabolised either directly to formate and carbon dioxide, or through the tetrahydrofolate-mediated pathways of one-carbon metabolism. However since the key intermediate, N-hydroxymethylnornicotine, could not be detected, the possibility of a direct methyl group transfer to tetrahydrofolate cannot be excluded.

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

“…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.…”

“…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.…”

“…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.Based on the available information concerning nicotine biosynthesis and N-demethylation in tobacco, one would expect that impaired MTHFR function in tobacco plants would reduce the production of Met and AdoMet by reducing 5-methyl-THF availability and that reduced AdoMet availability would limit nicotine biosynthesis in roots. In addition, if the methyl groups released from nicotine are recycled back to the THF-mediated C1 metabolic pathway in a regulated manner in response to a perceived C1 deficiency, then reduced MTHFR expression in tobacco plants may stimulate nicotine N-demethylation as a mechanism to replenish the THF-mediated C1 pool in leaves.…”

“…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.…”

“…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.…”