Monoterpene double-bond reductases of the (−)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (−)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (−)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (−)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (−)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and Km values of 1.0 and 2.2 μM for (−)-isopiperitenone and NADPH, respectively, with kcat of 1.3 s−1 for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (−)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and Km values of 2.3 and 6.9 μM for (+)-pulegone and NADPH, respectively, with kcat of 1.8 s−1. Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (−)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.