Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene.
(+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone. Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan. An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source. The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil.
Bertea
C M
CM
Department of Plant Biology, University of Turin, viale P.A. Mattioli 25, I-10125Turin, Italy.
Schalk
M
M
Karp
F
F
Maffei
M
M
Croteau
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000081005:Cyclohexane Monoterpenes; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D019686:Lamiaceae / Q000201:enzymology* / Q000378:metabolism; D008610:Menthol / Q000031:analogs & derivatives / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D039821:Monoterpenes; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013729:Terpenes / Q000378:metabolism*; D014162:Transfection
D000081005:Cyclohexane Monoterpenes; D018076:DNA, Complementary; D039821:Monoterpenes; D013729:Terpenes; D008610:Menthol; C054487:menthofuran; C039648:pulegone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C430094:menthofuran synthase