The search for useful drugs of defined structure from plants began with the isolation of morphine from dried latex, or opium, of the opium poppy Papaver somniferum in 1806 ( Sertürner 1806). The narcotic analgesic morphine and the antitussive and narcotic analgesic codeine, the antitussive and apoptosis inducer noscapine ( Ye et al. 1998 ), and the vasodilator papaverine are currently the most important physiologically active alkaloids from opium poppy. Of these four alkaloids, only papaverine is prepared by total chemical synthesis for commercial purposes. Opium poppy therefore serves as one of the most important renewable resources for pharmaceutical alkaloids. Per annum, 90–95% of the approximately 160 tons of morphine that are purified are chemically methylated to codeine, which is then used either directly or is further converted to a variety of derivatives such as dihydrocodeinone and 14-hydroxydihydrocodeinone that find use as antitussives and analgesics ( Kutchan 1998). The elicit production of morphine for acetylation to heroin is unfortunately almost 10 times that amount, more than 1200 tons per year ( Zenk 1994).
The enzymatic synthesis of morphine in opium poppy has been almost completely elucidated by M.H. Zenk and co-workers and is summarized by Kutchan (1998). Opium poppy produces more than 100 different alkaloids that are derived from the amino acid l-tyrosine and have the tetrahydrobenzylisoquinoline alkaloid (S)-reticuline as a common intermediate. There are three NADPH-dependent reductases involved in the conversion of (S)-reticuline to morphine. (S)-Reticuline must first be converted to (R)-reticuline before the phenanthrene ring with the correct stereochemistry at C-13 can be formed. The inversion of stereochemistry at C-1 of (S)-reticuline occurs by oxidation to the 1,2-dehydroreticulinium ion followed by stereospecific reduction to the R-epimer by 1,2-dehydroreticulinium ion reductase ( EC126.96.36.199) ( De-Eknamkul & Zenk 1992). The second reduction occurs after formation of the phenanthrene nucleus with stereospecific reduction of salutaridine to salutaridinol by salutaridine reductase ( EC188.8.131.52) ( Gerardy & Zenk 1993). The third reduction is the penultimate step in the biosynthetic pathway to morphine, the reduction of codeinone to codeine by codeinone reductase ( EC184.108.40.206) ( Fig. 1) ( Lenz & Zenk 1995a; Lenz & Zenk 1995b). The substrate for codeinone reductase, codeinone, exists in an equilibrium with its positional isomer neopinone. In vitro, as codeinone is reduced, this equilibrium is continually driven from neopinone towards codeinone until the substrates are depleted ( Gollwitzer et al. 1993 ). Each of the known enzymes of morphine biosynthesis has been detected in both P. somniferum plants and cell suspension culture, yet plant cell cultures have never been shown to accumulate morphine ( Kutchan 1998).
To date, none of the genes specific to morphine biosynthesis in opium poppy have been isolated. Tyrosine/dopa decarboxylase and a cytochrome P-450 reductase have been investigated at the molecular genetic level, but are involved in multiple biochemical processes in this plant (Facchini & De Facchini & Luca 1994 ; Rosco et al. 1997 ). Morphine, along with the chemotherapeutic agents vincristine, vinblastine and camptothecin, is one of the most important alkaloids commercially isolated from medicinal plants. Isolation of the genes of morphine biosynthesis would facilitate metabolic engineering of opium poppy to produce plants with specific patterns of alkaloids and could ultimately lead to an understanding of the inability of plant cell cultures to accumulate morphine. In this paper, we take the first step towards both of these goals with the isolation and characterization of cDNAs and genes that encode codeinone reductase isoforms in opium poppy. To our knowledge, this is the first report of the cloning of genes specific to morphine biosynthesis.