Biochemical Characterization of a Spearmint Mutant That Resembles Peppermint in Monoterpene Content

Abstract

A radiation-induced mutant of Scotch spearmint (Mentha x graclis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the CS-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and CS-oxygenated families of monoterpenes from the common precursor (-)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (-)-limonene hydroxylase; the C6-hydroxylase producing (-)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (--trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the ai-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3-and CS-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha.The monoterpene constituents of the essential oils of peppermint (Mentha piperita L.) and spearmint (native = Mentha spicata L.; Scotch = Mentha x gracilis [28]) are distinguished by the position of oxygenation on the p-menthane ring3 (18,25). Peppermint produces almost exclusively monoterpenes bearing an oxygen function at C3, such as menthone and menthol, whereas spearmint species produce almost exclusively monoterpenes bearing an oxygen function at C6, typi-

Monoterpene Metabolism. Cloning, Expression, and Characterization of (−)-Isopiperitenol/(−)-Carveol Dehydrogenase of Peppermint and Spearmint

“…In spite of the significant differences in essential oil chemistry, the observation that both dehydrogenases of peppermint and spearmint appear to utilize (2)-trans-carveol and (2)-trans-isopiperitenol as substrates (Croteau et al, 1991), and the facts that spearmint is a parent of peppermint (Murray et al, 1972;Harley and Brighton, 1977) and that other genes of the two species are very similar (Lupien et al, 1999), suggested that the target monoterpenol dehydrogenases would also be very similar. Polyclonal antibodies prepared against the peppermint ISPD ( Turner and Croteau, 2004) were used for immunoblotting of secretory cell extracts.…”

“…(2)-Carveol was used as substrate in the initial screen, because it is more readily available than (2)-trans-isopiperitenol. Based on previous results (Croteau et al, 1991) …”

“…Thus, pSBET clones were used to transform E. coli BL21(DE3) cells (Invitrogen, Carlsbad, CA), which were grown to A 600 of 0.8 to 1.0 at 37°C. Approximately 4 h after induction with 1 mM isopropyl b-D-thiogalactopyranoside, 100 mM (2)-transcarveol was added (neat) and the mixture was allowed to incubate overnight at 28°C; (2)-carveol is commercially available and was used as test substrate based on previous evidence that this monoterpenol was used by both peppermint and spearmint dehydrogenases (Croteau et al, 1991). At the completion of incubation, cultures were steam distilled-pentane extracted using a Likens-Nickerson apparatus (J&W Scientific, Folsum, CA) that was equipped with standard condenser cooled with chilled glycol.…”

“…Although the full complement of redox enzymes and the isomerase are present in spearmint (Croteau et al, 1991), (2)-carvone is an inefficient substrate for the initial double-bond reductase, thereby allowing (2)-carvone to accumulate. Thus, the oxidation of the initially formed monoterpenol is the only common redox step in monoterpene metabolism of these two Mentha species.…”

Molecular Cloning, Functional Expression and Characterization of d-Limonene Synthase from Agastache rugosa.

“…The major component, pulegone, was isolated by silica gel chromatography and determined to be the l-enantiomer based on its optical rotation. Since l-pulegone is supposed to be biosynthesized via d-limonene, 20) A. rugosa containing ubiquitous l-pulegone was expected to be a good source for cloning the d-limonene synthase gene. Using cDNA from A. rugosa as a template, a core fragment containing the 3Ј-flanking region of limonene synthase gene was obtained by PCR amplification with sense primer A, and subsequent 5Ј-RACE gave the 5Ј-terminal sequence.…”