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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Monoterpene Metabolism. Cloning, Expression, and Characterization of (–)-Isopiperitenol/(–)-Carveol Dehydrogenase of Peppermint and Spearmint¹

Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164–6340

The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (–)-trans-isopiperitenol to (–)-isopiperitenone in peppermint and (–)-trans-carveol to (–)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (–)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (–)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (–)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (–)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (–)-trans-isopiperitenol and (–)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (–)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.053298.

^* Corresponding author; e-mail croteau{at}mail.wsu.edu; fax 509–335–7643.

Received September 16, 2004; returned for revision December 17, 2004; accepted December 20, 2004.

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