Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves

Abstract

The monoterpene ketone l-menthone is specifically converted to I-menthol and I-menthyl acetate and to d-neomenthol and d-neomenthyl-f8-nglucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves. The selectivity of product formation results from compartmentation of the menthol dehydrogenase with the acetyl transferase and that of the neomenthol dehydrogenase with the glucosyl transferase. Soluble enzyme preparations, but not particulate preparations, from mature peppermint leaves catalyzed the NADPH-dependent reduction of 1-menthone to both epimeric alcohols, and the two dehydrogenases responsible for these stereospecific transformations were resolved by affinity chromatography on Mitrex Gel Red A. Both enzymes have a molecular weight of approximately 35,000, possess a Km for NADPH of about 2 x 10' M, are very sensitive to inhibition by thiol-directed reagents, and are not readily reversible. The menthol dehydrogenase showed a pH optimum at 7.5, exhibited a Km for 1-menthone of about 2.5 x 10' M, and also reduced disomenthone to d-neoisomenthol. The neomenthol dehydrogenase showed a pH optimum at 7.6, exhibited a K. for I-menthone of about 2.2 x 10' M, and also reduced d-isomenthone to d-isomenthol. These stereochemically distinct, but otherwise similar, enzymes are of key importance in determining the metabolic fate of menthone in peppermint, and they are probably typical of the class of dehydrogenases thought to be responsible for the metabolism of monoterpene ketones during plant development.appears to be a common feature of maturing essential oil plants. In peppermint, the metabolic disposition of the diastereomeric reduction products of 1-menthone is highly specific in that only 1-menthyl acetate (with little d-neomenthyl acetate) and d-neomenthyl-,f-D-glucoside (with little l-menthyl-fi-D-glucoside) are formed (6, 12). In vivo experiments, using labeled CO2, menthone, menthol and neomenthol as precursors, provided compelling evidence that compartmentation effects, and not the substrate specificity of the transferases, were responsible for the selectivity of pathways observed; this conclusion was confirmed by isolation and characterization of the acetyl CoA:monoterpenol acetyltransferase and the UDP-glucose:monoterpenol glucosyltransferase involved (5, 18). The nature of the terpenyl moiety of the acetate and ,B-D-glucoside is, thus, determined at the menthone reduction step by compartmentation of these stereospecific reactions with the appropriate, relatively nonselective, transferase. Consistent with chemical considerations (15) and with earlier genetic studies (13,19), such a metabolic scheme requires the presence in peppermint of two stereochemically distinct dehydrogenases. The NADPH-dependent conversion of d-pulegone to menthol (presumably via menthone) by extracts of peppermint leaves has been detected by Loomis and coworkers (3), suggesting the existence of one of the necessary enzymes. In this communication, we describe in detail the separation and characterization of t...

Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint

“…Substrates used were 180 m (Ϫ)-isopiperitenone (for isopiperitenone reductase) (Kjonaas et al, 1985), 200 m (ϩ)-cis-isopulegone (for cis-isopulegone isomerase) (Croteau and Venkatachalam, 1986), 200 m (ϩ)-pulegone (for pulegone reductase) , and 500 m (Ϫ)-menthone (for menthone reductase) (Kjonaas et al, 1982). All of the reductase assays also included 1 mm NADPH and an NADPH-regenerating system consisting of 1 unit of Glc-6-P dehydrogenase and 6 mm Glc-6-P.…”

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

“…Each of the assays outlined below, with the exception of the epoxidase assay, has been described in detail elsewhere (4,8,12,14,15). The assay for geranyl pyrophosphate:(-)-limonene cyclase depends upon the conversion of 1-3H-labeled precursor to the olefinic product, with TLC purification following the addition of authentic carrier and verification of product identity by radio-GLC (3,27 …”

“…The remaining enzymatic steps in the conversion of the first oxygenated products, (-)-transcarveol in spearmint and (-)-trans-isopiperitenol in peppermint, to carvone and menthol isomers, respectively ( Fig. 1), have also been previously described (4,8,14,15).…”

Development of Peltate Glandular Trichomes of Peppermint

“…1) have now shown that all but (Ϫ)-menthone reductase (Kjonaas et al, 1982) have a very similar developmental profile, with high levels of activity in leaves 12 to 20 d of age and a sharp peak of activity centered at 15 d . This correlation between in vitro enzyme activity and the rate of biosynthesis measured in vivo suggests that monoterpene formation is controlled by the coordinately regulated activity of the relevant biosynthetic enzymes.…”