Tetrahydrobenzylisoquinoline Alkaloid Biosynthesis

On the pathway leading from L-tyrosine to the first tetrahydrobenzylisoquinoline alkaloidal intermediate (S)-norcoclaurine, cDNAs encoding tyrosine/dopa decarboxylases (tydc) have been isolated (Fig. 10.5) (Facchini and De Luca, 1994). The transformation of (S)-norcoclaurine to the central isoquinoline alkaloid biosynthetic intermediate (S)-reticuline is quite well understood at both the enzyme and gene level (Fig. 10.6). (S)-Norcoclaurine is O-methylated at the 6-position by (R,S)-norcoclaurine 6-O-methyltransferase (6-omt) (Ru¨ ffer et al., 1983). cDNAs encoding this enzyme have been isolated fromThalictrum tuberosum, Coptis japonica, and P. somniferum (Frick and Kutchan, 1999; Morishige et al., 2000; Ounaroon et al., 2003). (S)- Coclaurine is next N-methylated by (R,S)-coclaurine N-methyltransferase (Frenzel and Zenk, 1990a). This cDNA has been characterized from C. japonica (Choi et al., 2002) and P. somniferum (S. Haase, J. Ziegler, S. Frick, and T. M. Kutchan, unpublished data). (S)-N-Methylcoclaurine is hydroxylated by the cytochrome P450 dependent monooxygenase cyp80b1 (S)-N-methylcoclaurine 3'-hydroxlyase (Pauli and Kutchan, 1998). The cDNA encoding this cytochrome P450 has been isolated from the California poppy Eschscholzia californica and from P. somniferum (Huang and Kutchan, 2000; Pauli and Kutchan, 1998). (S)-3'-Hydroxy-N-methylcoclaurine is methylated to (S)-reticuline by (R,S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-omt) (Frenzel and Zenk, 1990b). The cDNA 4'-omt has been isolated from C. japonica (Morishige et al., 2000) and from P. somniferum (Ziegler et al., 2005).

FIGURE 10.6 Schematic representation of the biosynthetic pathway leading from dopamine and p-hydroxyphenylacetaldehyde to laudanine. 6-omt, (R,S)-norcoclaurine 6-O-methyltransferase; NMT, (R,S)-coclaurine, N-methyltransferase; cyp80b1, (S)-N-methylcoclaurine 3'-hydroxylase; 4'-omt, (R,S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; 7-omt, (R,S)-reticuline 7-O-methyltransferase.

(S)-Reticuline is the chemical chameleon of isoquinoline alkaloid biosynthesis, which can lead to a plethora of alkaloidal structures. In P. somniferum, (R,S)-reticuline can be methylated by (R,S)-reticuline 7-O-methyltransferase, for which the cDNA 7-omt has been described, to the tetrahydrobenzylisoquinoline laudanine (Fig. 10.6 Ounaroon et al., 2003). Along the pathway in which (S)-reticuline is specifically converted to morphine, cDNAs encoding two biosynthetic enzymes have been identified (Fig. 10.7). Salutaridinol 7-O-acetyltransferase, encoded by SalAT, transfers an acetyl moiety from acetyl-CoA to the 7-hydroxyl group of salutaridinol (Grothe et al., 2001; Lenz and Zenk, 1995a). Codeinone reductase is encoded by cor1 and catalyzes the penultimate step in morphine biosynthesis, the NADPH-dependent reduction of the keto moiety of codeinone to the 6-hydroxyl group of codeine (Lenz and Zenk, 1995b; Unterlinner et

FIGURE 10.7 Schematic representation of the biosynthetic pathway leading from (S)-reticuline to morphine. SalAT, salutaridinol 7-O-acetyltransferase; COR1, codeinone reductase.

al., 1999).

In P. somniferum and E. californica, the N-methyl group of (S)-reticuline can be oxidatively cyclized by the bbe to the bridge carbon, C-8, of (S)-scoulerine (Fig. 10.8; Rink and Böhm, 1975; Steffens et al., 1985). (S)-Scoulerine is then further converted in these plants to antimicrobial benzo[c]phenathridine alkaloids, such as sanguinarine. cDNAs encoding the bbe have been isolated from E. californica, P. somniferum, and Berberis stolonifera (Chou and Kutchan, 1998; Dittrich and Kutchan, 1991; Facchini et al., 1996; Huang and Kutchan, 2000). (S)-Reticuline is converted via (S)-scoulerine to berberine alkaloids in Berberis and Coptis species. Along the biosynthetic pathway to berberine, two cDNAs have been identified from C. japonica. (S)-Scoulerine is methylated by (S)-scoulerine 9-O-methyltransferase (9-omt) (Muemmler et al., 1985;

FIGURE 10.8 Schematic representation of the biosynthetic pathway leading from (S)-reticuline to sanguinarine and berberine. bbe, berberine bridge enzyme; 9-omt, (S)-scoulerine 9-O-methyltransferase; CYP719, (S)-canadine synthase.

Takeshita et al., 1995) to (S)-tetrahydrocolumbamine which is subsequently acted upon by CYP719 (Bauer and Zenk, 1991; Ikezawa et al., 2003; Rueffer and Zenk, 1994), a cytochrome P450- dependent enzyme that catalyzes formation of the methylenedioxy bridge of (S)-canadine.

With the current collection of cDNAs encoding enzymes of tetrahydrobenzylisoquinoline alkaloid biosynthesis, some progress has also been made with respect to our understanding of the spatial regulation of this biosynthesis. The cellular localization of tetrahydrobenzylisoquinoline alkaloid biosynthesis will next be considered.