Molecular Genetic Analysis of an O-Methyltransferase of the Opium Poppy Papaver somniferum

Isoquinoline alkaloids are a large class of compounds derived from the amino acid L-tyrosine containing many physiologically active members. Among the isoquinoline alkaloids, morphine is one of the pharmaceutically important members that is still derived from the plant that produces it, the opium poppy Papaver somniferum. P. somniferum produces over 80 alkaloids derived from L-tyrosine. We have isolated cDNAs encoding several enzymes of tetrahydrobenzylisoquinolinederived alkaloid biosynthesis from this plant. The first enzyme in the biosynthetic pathway for which we have isolated a cDNA is norcoclaurine 6-O-methyltransferase. The next is the cytochrome P-450-dependent monooxygenase (S)-Nmethylcoclaurine 3’-hydroxylase. These enzymes are common to the morphine, noscapine and sanguinarine biosynthetic pathways. Specific to the sanguinarine pathway is the berberine bridge enzyme that oxidatively cyclizes the N-methyl moiety of (S)-reticuline to the bridge carbon C-8 of (S)-scoulerine. Finally, specific to morphine biosynthesis are salutaridinol 7-O-acetyltransferase and codeinone reductase the penultimate enzyme of the morphine pathway that reduces codeinone to codeine. Given the number of cDNAs specific to various alkaloid biosynthetic pathways that we now have, attempts at metabolic engineering of P. somniferum can be made. We describe herein details of the isolation and biochemical characterization of a cDNA encoding the P. somniferum O-methyltransferase OMTPS3. INTRODUCTION Alkaloids are pharmacologically active, nitrogen-containing, basic compounds originally believed to be only of plant origin. Since the isolation of the first alkaloid morphine, more than 12,000 alkaloids have been defined. Approximately 20% of flowering plants produce alkaloids. Each species accumulates alkaloids in a unique and defined patterned. The role of alkaloids in plants has been a longstanding question, but a picture emerges that supports an ecochemical function for these compounds. Alkaloidcontaining plants were also mankind’s original materia medica. Many of these plants are still used today as sources of prescription drugs, for example the analgesics morphine and codeine are isolated from the opium poppy Papaver somniferum. Alkaloid biosynthetic pathways are attractive targets for molecular biology because of their role in plant chemical ecology and the biotechnological potential for the production of commercially important compounds. S-Adenosyl-L-methionine-dependent methyltransferases are involved in the Omethylation of many plant natural products. For several alkaloids of the isoquinoline type, the complete biosynthetic pathway has been elucidated at the enzyme level, which provides us with insightful information into the substrate specificity of the various biosynthetic enzymes (reviewed in Kutchan, 1998). The biosynthesis of the central isoquinoline alkaloid intermediate (S)-reticuline requires the action of two O-methyltransferases, norcoclaurine 6-O-methyltransferase and 3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase (Rüffer et al., 1983; Frenzel and Zenk, 1990; Sato et al., 1994; Hara et al., 1995). Partial purification and characterization of these enzymes indicated that they might have relatively strict substrate specificity. It was thought that only minor Proc. WOCMAP III, Vol. 1: Bioprospecting & Ethnopharmacology Eds. J. Bernáth, É. Németh, L.E. Craker and Z.E.Gardner Acta Hort 675, ISHS 2005 168 variations to the presumed in vivo substrate could be tolerated by the enzymes. This was found not only to be true for alkaloid biosynthetic pathways, but for other types of natural products as well. Regiospecific oxygen methylation significantly contributes to the vast metabolic diversity of plant secondary metabolism. O-methyltransferase encoding genes are tentatively identified and annotated based upon sequence similarity to those of other proteins (Ibrahim et al., 1998; Joshi and Chiang, 1998; Schröder et al., 2002). Understanding gene function, however, requires more substantial biochemical characterization. An example of how we approach this problem with respect to methyltransferases is provided herein. MATERIALS AND METHODS Plant Material P. somniferum seedlings were routinely grown aseptically on Gamborg B5 medium (Gamborg et al., 1968) containing 0.8% agar in a growth chamber at 22°C, 60% relative humidity under cycles of 16 h light/8 h dark with a light intensity of 85 μmol sec m per μA. Differentiated P. somniferum plants were grown either outdoors in Saxony-Anhalt or in a greenhouse at 24°C, 18 h light and 50% humidity. Generation of Partial cDNAs from P. somniferum Partial cDNAs encoding O-methyltransferases from P. somniferum were produced by PCR using cDNA generated by reverse transcription of mRNA isolated from floral stem. DNA amplification using either Taq or Pfu polymerase was performed under the following conditions: 3 min at 94°C, 35 cycles of 94°C, 30 s; 50°C, 30 s; 72°C, 1 min. At the end of 35 cycles, the reaction mixtures were incubated for an additional 7 min at 72°C prior to cooling to 4°C. The amplified DNA was resolved by agarose gel electrophoresis, the band of approximately correct size (400 bp) were isolated and subcloned into pGEMT Easy (Promega) prior to nucleotide sequence determination. Generation of Full-Length cDNAs The complete nucleotide sequence was generated in two steps using one Omethyltransferase-specific PCR primer and one RACE-specific primer as specified by the manufacturer. The 5’and 3’-RACE-PCR experiments were carried out using a SMART cDNA amplification kit (Clontech). RACE-PCR was performed using the following PCR cycle: 3 min at 94°C, 25 cycles of 94°C, 30 s; 68°C, 30 s; 72°C, 3 min. At the end of 25 cycles, the reaction mixtures were incubated for an additional 7 min at 72°C prior to cooling to 4°C. The amplified DNA was resolved by agarose gel electrophoresis, the band of the expected size were isolated and subcloned into pGEM-T Easy prior to sequencing. The full-length clone was generated in one piece using primers specific for the open reading frame for PCR with P. somniferum floral stem cDNA as template. The final primers used for cDNA amplification contained recognition sites for the restriction endonucleases BamHI and NotI, appropriate for subcloning into pFastBac HTa (Life Technologies) for functional expression. DNA amplification was performed under the following conditions: 3 min at 94°C, 35 cycles of 94°C, 30 s; 60°C, 30 s; 72°C, 2 min. At the end of 35 cycles, the reaction mixtures were incubated for an additional 7 min at 72°C prior to cooling to 4°C. The amplified DNA was resolved by agarose gel electrophoresis, the band of approximately correct size was isolated and subcloned into pCR4-TOPO (Invitrogen) prior to nucleotide sequence determination. Heterologous Expression and Enzyme Purification The full-length cDNA generated by RT-PCR was ligated into pFastBac HTa that had been digested with restriction endonucleases BamHI and NotI. The recombinant plasmid was transposed into baculovirus DNA in the Escherichia coli strain DH10BAC (Life Technologies) and then transfected into Spodoptera frugiperda Sf9 cells according to the manufacturer’s instructions. The insect cells were propagated and the recombinant