The 1-benzyltetrahydroisoquinoline (BTIQ) skeleton contains a tetrahydroisoquinoline (TIQ) segment and a benzyl-derived moiety. According to Winterstein and Trier the BTIQ nucleus is formed by Picket—Spencer condensation of dopamine with 3,4- dihydroxyphenylacetaldehyde (dopal). Tracer experiments indicated that (S)-nor- laudanosoline (tetrahydroxylated BTIQ) is probably the first intermediate in the biosynthesis of morphinanes (Battersby et al., 1965). Tyrosine has been found to label both the TIQ and benzylic parts of morphinanes (Battersby et al., 1962; Neubauer, 1965; Roberts et al., 1987). When [1-13C]tyramine or [2-14C]tyramine were infiltrated into Papaver somniferum plants (Roberts et al., 1987) and [2- 13C]tyramine into developing poppy seedlings (Loeffler et al., 1987), only the TIQ portion of morphine was labelled. These findings indicate that tyramine is not a precursor of the benzylic part of the BTIQ ring system. After administration of labelled dopa and dopamine only the TIQ segment of alkaloids was labelled (Spencer, 1968). These results demonstrated that in Papaver somniferum tyramine is not transformed by amine oxidase into 4-hydroxyphenylacetaldehyde (tyral) as reported in Berberis cell cultures (Rueffer and Zenk, 1987). Highly enriched thebaine and morphine labelled specifically at position 9 were obtained after infiltration of (R, S)-[1-13C]norcoclaurine and (R, S)[1-13C]coclaurine into opium poppy seedlings (Loeffler et al., 1987). Precursor feeding experiments to Annona reticulata leaves demonstrated that coclaurine and reticuline are both derived from norcoclaurine (Stadler et al., 1987). These results indicated that not tetrahydroxylated norlaudanosoline but trihydroxylated norcoclaurine is the first BTIQ intermediate in the biosynthesis of opium poppy alkaloids. Feeding experiments with (S)-[l-13C]norcoclaurine showed that norcoclaurine is specifically incorporated into protoberberine, berberine, aporphine, benzophenathridine and pavine alkaloids (Stadler et al., 1987, 1989; Müller and Zenk, 1992). If norcoclaurine is the first BTIQ intermediate in the biosynthesis of 1-benzylisoquinoline alkaloids, than the carbonyl-condensation unit is 4-hydroxyphenylacetaldehyde (tyral). Tyral can arise by decarboxylation of p-hydroxyphenylpyruvate, formed by transamination from tyrosine (Rueffer and Zenk, 1987).
The proposed biochemical relationships at the interface between shikimate and norcoclaurine biosynthesis are schematically shown in Figure 5.
Transaminase, phenoloxidase, decarboxylase and oxidative deaminase activities have been reported to occur in opium poppy plants (Jindra et al., 1966; Kovács and Jindra, 1965; Kovács, 1970). The alkaloids of opium poppy have been identified in the alkaloid vesicles of latex (Fairbairn et al., 1974). Later on, phenoloxidase activity (Roberts, 1971, 1974) and its bound and soluble forms (Antoun and Roberts, 1975), transaminase and decarboxylase activities (Antoun and Roberts, 1975; Roberts and Antoun, 1978; Roberts et al., 1983) were studied in Papaver somniferum latex. A specific hydroxylation of tyramine was reported by phenoloxidase from opium poppy plants (Asghar and Siddiqi, 1969).
Immobilized cells of Papaver somniferum retained their tyrosine/dopa decarboxylase activity for several months (Stano et al., 1995).
The enzymes involved in the transformation of tyrosine to dopamine and tyral have been identified and characterized partially in opium poppy. Neither of these enzymes was purified to homogeneity and their molecular characteristics have not been investigated (Hashimoto and Yamada, 1994). Molecular analyses of these Figure 5 The enzymes in the biosynthesis of dopamine and tyral in BTIQ-alkaloid forming plants 1—phenoloxidase, 2—decarboxylase, 3—transaminase, 4—amine oxidase, 5— norcoclaurine synthase
enzymes and the corresponding genes have begun recently in order to gain a better understanding of the regulation of carbon flow between shikimate and BTIQ alkaloid biosynthetic pathways.
4.1 Tyrosine/3,4-dihydroxyphenylalanine Decarboxylase
Primers designed to two conserved domains of other aromatic amino acids decarboxylases have been used for PCR amplification of a poppy genomic DNA fragment and an insert from an authentic clone of tryptophan decarboxylase (TDC) from C. roseus (Facchini and De Luca, 1994). The TDC inserts and the PCR product were used to screen an opium poppy seedling cDNA library. Two different cDNAs (cTyDC2 and cTyDC3) for tyrosine/dopa decarboxylase have been identified by heterologous screening by TDC cDNA as probe and a third independent cTyDC1 was detected by the PCR fragment from opium poppy genomic DNA. Screening of a poppy genomic library by cTyDC1 resulted in the isolation of two genomic clones (gTyDC1 and gTyDC4). The nucleotide sequence of gTyDC1 was found to be identical to cTyDC1 and that of gTyDC4 was 90% identical to cTyDC1. Members of the poppy tyrosine/dopa decarboxylase gene family have been divided into two subsets (cTyDC1 and gTyDC4, cTyDC2 and cTyDC3) according to sequence homology. Within each subset the clones exhibit about 90% identity, whereas clones between subsets share less than 75% identity. One member of each subset (cTyDC1 and cTyDC2) was used to screen the poppy genomic DNA fragments obtained by digestion with four restriction endonucleases. Six to eight and four to six positive bands were found for each digestion by cTyDC1 and cTyDC2 respectively. These results showed that tyrosine/dopa decarboxylase is encoded in the opium poppy by a family of 10- 14 genes. When gTyDC1 and cTyDC2 were expressed in E. coli as ß-galactosidase fusion proteins, these proteins exhibited high substrate specificity for dopa and tyrosine. For these enzymes tryptophan and phenylalanine have not been accepted as substrates. The Km values of the enzymes were equal for both tyrosine and dopa (Km1 mM). Both enzymes exhibited a similar broad pH optimum (pH 7.5—8.5) (Facchini and De Luca, 1995a). By in situ hybridization using TyDC1 and TyDC2 sense and anti-sense probes, an analysis of the spatial distribution of TyDC transcripts showed their association with vascular tissue in mature roots and stems. For analysis of organ-specific expression (roots, stems, leaves, sepals, carpels, stamens and petals) samples were collected from mature poppy plants with fully expanded flowers. RNA blot-hybridization analysis with cTyDC1 and cTyDC2 as probes showed that the TyDC1-like transcripts were expressed predominantly in roots and the TyDC2-like transcripts were identified mostly in stems and roots (Facchini and De Luca, 1995b) and to a much lesser extent in other organs analysed. The levels of TyDC1- and TyDC2-like transcripts were similar along the stem length. The level of both transcripts was low in the basal parts of the stem. During carpel development (from floral bud through anthesis to maturation) the levels of TyDC1- and TyDC2-like transcripts were detectable only up to anthesis. After pollination, in expanding carpels, the level of these transcripts was undetectable.
The content of thebaine and codeine was found to be constantly low in latex from expanding carpels (Zichová et al., 1996). The level of morphine approximately
doubled in latex collected from expanding capsules (from pollination up to the eighth day) and remained almost constant in the next stages of capsule maturation. In latex collected from expanding carpels and during their maturation the content of tyrosine is constantly low, but the level of tyramine, and particularly that of dopamine, is high, reaching a maximum at the highest content of morphine. Whether morphine is transported into the expanding carpels or morphine is de novo formed in these carpels from substrates present in the latex remains to be clarified.
Another member of the TyDC/DoDC gene family, TyDC5, has been isolated from a genomic library of opium poppy (Maldonado-Mendoza et al., 1996). When the coding region of TyDC5 was expressed in E. coli the highest recombinant enzyme activity was with tyrosine and dopa (only 64% of that with tyrosine) and an extremely low activity was detected with phenylalanine. No activity was observed against tryptophan. The organ-specific expression of TyDC5 has been analysed by ribonuclease protection assay. TyDC5 has been expressed only in the roots of mature poppy plants. In other organs, including the latex and cell suspension cultures, TyDC5 expression was almost undetectable. A high expression of this gene has been detected in developing poppy seedlings. When a promoter fraction of TyDC5 was fused to GUS reporter gene, the expression of TyDC5::GUS gene construct expression was restricted to the roots in transgenic tobacco plants, similarly as determined by ribonuclease protection assay in roots of poppy plants. The deduced amino acid sequence and root-specific expression pattern of TyDC5 are most similar to those of TyDC1 (Facchini and De Luca, 1995a, b), suggesting that they are members of a common TyDC/DoDC gene family.