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...