Biochemical and molecular characterizations of nicotine demethylase in tobacco

Plant cytochrome P450 (CYP) enzymes are involved in the biosynthesis of many primary and secondary metabolites including phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates. However, while hundreds of CYP genes have been identified in plant genomes, relatively few have been functionally characterized. We report here the cloning and characterization of a CYP enzyme from tobacco (Nicotiana tabacum L.) that demethylates nicotine to form nornicotine, a precursor to the nitrosamine N′-nitrosonornicotine (NNN). Microsomal demethylase activity was first shown to be induced in leaves treated with the growth regulator ethylene, required molecular oxygen and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for full activity, and exhibited a K_m of 3.9 μM for nicotine and a maximum turnover rate (V_max) of 8.9 pkat mg ^-1 protein. Equally important, microsomal activity was reduced greatly by several inhibitors of CYP-mediated reactions. Using a polymerase chain reaction based strategy with degenerate primers designed to conserve P450 motifs and mRNA from ethylene-treated leaves, putative gene fragments representing 32 different P450 gene families were isolated and numerous full-length genes were cloned. Employing GeneChip® (Affymetrix Inc., Santa Clara, CA) microarray hybridizations, the steady-state mRNA level of two highly related full-length cDNAs and one cDNA fragment were demonstrated to be highly expressed in ethylene-treated vs. control plant material. Microsomes from yeast over-expressing the two full-length cDNAs along with two other highly homologous P450 genes demonstrated that only one cDNA, D121-AA8 (GenBank accession no. DQ205656) encoded for demethylation activity comparable to that found in planta. Molecular modeling was used to identify putative active site residues and for comparisons to other putative demethylase cDNAs.