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CORESTA Congress, Paris, 2006, IG 02

Functional characterization of the nicotine N-demethylase gene of tobacco

DEWEY R.E.; SIMINSZKY B.; BOWEN S.W.; GAVILANO L.; WAREK U.; HAYES A.
North Carolina State University, Dept. of Crop Science, Raleigh, NC, USA; University of Kentucky, Dept. of Plant and Soil Sci., Lexington, KY, USA; Philip Morris USA, Richmond, VA, USA

In tobacco, nicotine is metabolized to nornicotine via the activity of the nicotine N -demethylase enzyme. During the curing and processing of the leaf, nornicotine serves are the direct precursor in the synthesis of N -nitrosonornicotine (NNN), a tobacco-specific nitrosamine. Therefore, limiting the production of nornicotine within in the leaf represents a viable strategy for ensuring low NNN synthesis and accumulation in the final processed product. Maintaining low nornicotine levels, however, has been particularly problematic in Burley tobaccos because of the unstable nature of the genetic locus that regulates the conversion of nicotine to nornicotine, a process that leads to high proportions of progeny (termed converters) that produce unacceptably high amounts of nornicotine. We have recently utilized a microarray-based transcript profiling strategy to isolate the nicotine N -demethylase gene from tobacco. RNA interference-mediated inhibition of this gene proved to be very effective in suppressing the synthesis of nornicotine in both greenhouse and field grown transgenic plants. The nicotine N -demethylase gene product is a member of the cytochrome P450 superfamily of oxidative enzymes. In the course of our studies, it became apparent that the tobacco genome encodes several additional P450 genes that are very closely related to the nicotine N -demethylase gene (sharing over 90% sequence homology), yet do not encode enzymes capable of demethylating nicotine. We have conducted a series of domain swapping experiments, in conjunction with site-directed mutagenesis, to establish the critical residues that are specifically required for the enzyme to function as a nicotine N -demethylase. The efficacy of transgenic strategies to reduce nornicotine levels in tobacco along with insights regarding the molecular evolution of the nicotine N -demethylase gene will be presented.