Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers.

In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes.

Matrix-bound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in tobacco: quantification and evidence for an origin from lignin-incorporated alkaloids.

Substantial quantities of the carcinogenic tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (1; NNK) are still found in the mainstream smoke of tobacco exhaustively extracted with water, indicating the presence of an insoluble, matrix-bound form. Soluble and matrix-bound concentrations of 1 in tobacco were determined by applying a new method using sequential aqueous extraction at room temperature and at 130 °C. On average, 77% and 53% of the total content of 1 were matrix-bound in air-cured (Burley type) and flue-cured tobaccos, respectively.