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First published online February 22, 2005; 10.1104/pp.104.053306 Plant Physiology 137:873-881 (2005) © 2005 American Society of Plant Biologists Monoterpene Metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint1Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164–6340
(–)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (–)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(–)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (–)-(3R)-menthol from (–)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and Km values of 674 µM, > 1 mM, and 10 µM for menthone, isomenthone, and NADPH, respectively, with a kcat of 0.06 s–1. The recombinant menthone:(–)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (–)-menthone to 95% (–)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has Km values of 3.0 µM, 41 µM, and 0.12 µM for menthone, isomenthone, and NADPH, respectively, with a kcat of 0.6 s–1. The respective activities of these menthone reductases account for all of the menthol isomers found in the essential oil of peppermint. Biotechnological exploitation of these genes could lead to improved production yields of (–)-menthol, the principal and characteristic flavor component of peppermint.
1 This work was supported by the U.S. Department of Energy, by the Mint Industry Research Council, and by the Washington State University Agricultural Research Center (project 0268). 2 Present address: Department of Biomedical Sciences, Tufts University, School of Veterinary Medicine, 200 Westboro Rd., North Grafton, MA 01536. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.053306. * Corresponding author; e-mail croteau{at}wsu.edu; fax 509–335–7643. Received September 16, 2004; returned for revision December 17, 2004; accepted December 20, 2004. This article has been cited by other articles:
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