TY - JOUR
T1 - Enantioselective demethylation of nicotine as a mechanism for variable nornicotine composition in tobacco leaf
AU - Cai, Bin
AU - Siminszky, Balazs
AU - Chappell, Joseph
AU - Dewey, Ralph E.
AU - Bush, Lowell P.
PY - 2012/12/14
Y1 - 2012/12/14
N2 - 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 (EFnnic 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.
AB - 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 (EFnnic 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.
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U2 - 10.1074/jbc.M112.413807
DO - 10.1074/jbc.M112.413807
M3 - Article
C2 - 23100254
AN - SCOPUS:84871125625
SN - 0021-9258
VL - 287
SP - 42804
EP - 42811
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 51
ER -