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 (Me ntha x piperita) essential oil that is derived from the alpha,beta -unsatur ated ketone (+)-pulegone. Microsomal preparations, from the oil gland secre tory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium , transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and mo lecular oxygen, implying that menthofuran is synthesized by a mechanism ana logous to that of mammalian liver cytochrome P450s involving the hydroxylat ion of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyc lization to the hemiketal, and dehydration to the furan, An abundant cytoch rome P450 clone from a peppermint oil gland cell cDNA library was functiona lly expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). Th e 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 anti cipated primary structural elements of a cytochrome P450 and most closely r esembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limo nene-3-hydroxylase, from the same source, The availability of this gene per mits transgenic manipulation of peppermint to improve the quality of the de rived essential oil. (C) 2001 Academic Press.