title: Enantioselective demethylation of nicotine as a mechanism for variable nornicotine composition in tobacco leaf.
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.
authors.affiliation: Department of Plant and Soil Sciences,University of Kentucky, Lexington, Kentucky 40546-0312, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Cai
authors.forename: Balazs
authors.initials: B
authors.lastname: Siminszky
authors.forename: Joseph
authors.initials: J
authors.lastname: Chappell
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Dewey
authors.forename: Lowell P
authors.initials: LP
authors.lastname: Bush
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis / Q000737:chemistry; D053898:Biosynthetic Pathways; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008954:Models, Biological; D009538:Nicotine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D010940:Plant Proteins; D009538:Nicotine; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System; C516919:nicotine demethylase, tobacco