Demonstration That Menthofuran Synthase of Mint (Mentha) Is a Cytochrome P450 Monooxygenase: Cloning, Functional Expression, and Characterization of the Responsible Gene 1 Cinzia M. Bertea,* ,2 Michel Schalk,† ,2,3 Frank Karp,† Massimo Maffei,* and Rodney Croteau† ,4 *Department of Plant Biology, University of Turin, viale P.A. Mattioli 25, I-10125 Turin, Italy; and †Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 Received February 12, 2001, and in revised form March 21, 2001; published online May 23, 2001 ()-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha  piperita) essen- tial oil that is derived from the ,-unsaturated ketone ()-pulegone. Microsomal preparations, from the oil gland secretory cells of a high ()-menthofuran-pro- ducing chemotype of Mentha pulegium, transform ()- pulegone to ()-menthofuran in the presence of NADPH and molecular oxygen, implying that mentho- furan is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of ()-pule- gone, 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 Sac- charomyces cerevisiae and Escherichia coli and shown to encode the ()-menthofuran synthase (i.e., ()-pule- gone-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 hydroxy- lase, ()-limonene-3-hydroxylase, from the same source. The availability of this gene permits trans- genic manipulation of peppermint to improve the quality of the derived essential oil. © 2001 Academic Press Key Words: monoterpene biosynthesis; ()-mentho- furan; ()-pulegone; cytochrome P450 monooxygen- ase; menthofuran synthase; pulegone hydroxylase; Mentha  pulegium; Mentha  piperita. The monoterpenoid (R)-(+)-menthofuran (Fig. 1) is a common component of the essential oil of several Men- tha (mint; Lamiaceae family) species, including pep- permint, in which levels in excess of a few percent are considered to decrease to quality of the distilled com- mercial product (1, 2). The accumulation of menthofu- ran is highly dependent on environmental conditions, with high day temperatures, high night temperatures, low photon flux densities, short days, and water stress resulting in the increased production of this metabolite (3, 4). That (R)-(+)-menthofuran is derived from (R)- (+)-pulegone (Fig. 1) has been demonstrated by in vivo feeding studies with peppermint (5, 6), but little else is known about this biosynthetic transformation in plants. The mammalian metabolism of the abortifacient ter- pene (R)-(+)-pulegone (usually ingested in the form of pennyroyal (Mentha pulegium) oil) has been studied extensively because of the often severe toxic reactions associated with the unregulated use of this herbal rem- edy (7–9). The conversion of (R)-(+)-pulegone to (R)- (+)-menthofuran as a proximate mammalian hepa- toxin is of pharmacological significance (10 –12) and has been shown to be mediated by human liver cyto- 1 This investigation was supported in part by a National Science Foundation grant, a grant from the Mint Industry Research Council, and by Project 0268 from the Agricultural Research Center, Wash- ington State University (to R.C.). C.M.B. was supported in part by a Graduate Fellowship in Biochemical Sciences, University of Turin, Italy. The nucleotide sequence reported in this paper has been sub- mitted to the GenBank/EMBL Data Bank under Accession No. AF346833. 2 The first two authors share equal credit in the research. 3 Present address: Firmenich SA, Route de Jeunes 1, CH-1211 Geneve 8, Switzerland. 4 To whom correspondence and reprint requests should be ad- dressed. Fax: (509) 335-7643. E-mail: croteau@mail.wsu.edu. 0003-9861/01 $35.00 279 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 390, No. 2, June 15, pp. 279 –286, 2001 doi:10.1006/abbi.2001.2378, available online at http://www.idealibrary.com on