Evidence for the involvement of tetrahydrofolate in the demethylation of nicotine by Nicotiana plumbaginifolia cell-suspension cultures.
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.
Mesnard
François
F
Laboratoire de Phytotechnologie EA 2085, Faculté de Pharmacie, Université de Picardie, 1 Rue des Louvels, 80037 Amiens Cedex 1, France.
Roscher
Albrecht
A
Garlick
Andrew P
AP
Girard
Sandrine
S
Baguet
Evelyne
E
Arroo
Randolf R J
RR
Lebreton
Jacques
J
Robins
Richard J
RJ
Ratcliffe
GeorgeR
G
D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008956:Models, Chemical; D009538:Nicotine / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010936:Plant Extracts / Q000032:analysis; D012694:Serine / Q000378:metabolism; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism*
D002250:Carbon Radioisotopes; D010936:Plant Extracts; D013763:Tetrahydrofolates; D006710:Homocysteine; C030371:5,6,7,8-tetrahydrofolic acid; D012694:Serine; D009538:Nicotine; D008715:Methionine; D003545:Cysteine