Abstract

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2'-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF(nnic) of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

Highlights

  • Nornicotine composition in tobacco leaf has been a puzzle for more than half a century

  • The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta

  • CYP82E5v2 and CYP82E10 account for Ͻ5% of the nicotine demethylation observed in plants that accumulate high levels of nornicotine, they are responsible for the majority of the nornicotine found in plants with a nonactivated or mutant CYP82E4 gene [6]

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Summary

Introduction

Nornicotine composition in tobacco leaf has been a puzzle for more than half a century. Conclusion: Nornicotine composition in tobacco leaf can be reasonably explained by the combination of three nicotine demethylases. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ϳ0.2% of the total nicotine pool. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf

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