Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint

Abstract

(-)-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 K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) 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 K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) 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.

Figure 1.

The primary pathway for monoterpene biosynthesis in peppermint. The enzymes responsible for the transformations shown are GPPS (1), (−)-limonene synthase (2), (−)-limonene-3-hydroxylase (3), (−)-trans-isopiperitenol dehydrogenase (4), (−)-isopiperitenone reductase (5), (+)-cis-isopulegone isomerase (6), (+)-menthofuran synthase (7), (+)-pulegone reductase (8), (−)-menthone:(−)-(3R)-menthol reductase (9), and (−)-menthone:(+)-(3S)-neomenthol reductase (10).

Figure 2.

Alignment of the deduced amino acid sequences of the (−)-menthone:(−)-(3R)-menthol reductase (MMR) and (−)-menthone:(+)-(3S)-neomenthol reductase (MNR), isopiperitenone reductase (ISPR) and (−)-trans-isopiperitenol dehydrogenase (ISPD) of peppermint. Highly conserved sequences (between MNR and ISPR) used to synthesize primers for the RT-PCR-RACE-based cloning of MMR are overscored by bars and the conserved motifs discussed in the accompanying paper (Ringer et al., 2005) are underlined by bars. Identical residues and similar residues are shaded black and gray, respectively.

Figure 3.

Gas chromatographic analyses of monoterpenol formation by recombinant MMR with menthone (B) and isomenthone (D) as substrates, and by recombinant MNR with menthone (C) and isomenthone (E) as substrates. The monoterpene standards (A) are menthone (1), isomenthone (2), neomenthol (3), neoisomenthol (4), menthol (5), and isomenthol (6). Assays for the conversion of isomenthone to monoterpenols (D and E) were scaled up 12-fold in time and 16-fold in protein concentration to increase product yield to that comparable for the conversions of menthone to monoterpenols (B and C).