Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum.

Salutaridinol 7-O-acetyltransferase (EC ) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K(m) value for salutaridinol was determined to be 9 microm and 54 microm for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3'-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.

The opium poppy Papaver somniferum produces some of the most widely used medicinal alkaloids. The narcotic analgesic morphine and the antitussive and narcotic analgesic codeine are the most important physiologically active alkaloids from this plant. Nineteen total syntheses of morphine have been reported through 1999 (1). The most efficient synthesis of morphine proceeded on medium scale with an overall yield of 29% (2). Despite many years of excellent synthetic organic chemistry concentrated on morphinans, a commercially feasible total chemical synthesis has not yet been achieved for morphine or codeine.
The enzymatic synthesis of morphine in P. somniferum has been almost completely elucidated by M. H. Zenk and co-workers and is summarized by Kutchan (3). Morphine is derived from two molecules of the amino acid L-tyrosine in a series of at least 17 enzymatic steps. The latter steps in the pathway that lead specifically from (S)-reticuline, a central intermediate of isoquinoline alkaloid biosynthesis, to morphine involve three NADPH-dependent oxidoreductases (4 -6), most probably three cytochromes P-450 (7), and an acetyl-CoA-dependent acetyltransferase (8).
Acetyl-CoA-dependent acetyltransferases have an important role in plant alkaloid metabolism. They are involved in the synthesis of monoterpenoid indole alkaloids in medicinal plant species such as Rauwolfia serpentina. In this plant the enzyme vinorine synthase transfers an acetyl group from acetyl-CoA to 16-epi-vellosimine to form vinorine. This acetyl transfer is accompanied by a concomitant skeletal rearrangement from the sarpagan to the ajmalan type (9). An acetyl-CoA-dependent acetyltransferase also participates in vindoline biosynthesis in Catharanthus roseus, the source of the chemotherapeutic dimeric indole alkaloid vinblastine (10,11). Acetyl-CoA:deacetylvindoline 4-O-acetyltransferase catalyzes the last step in vindoline biosynthesis. Central to morphine biosynthesis in P. somniferum is acetyl-CoA:salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) (Fig. 1). Acetylation of the phenanthrene salutaridinol is followed by allylic syn-displacement of the acetylated (activated) hydroxyl by the phenolic hydroxyl, which follows stereocontrol for S N 2Ј substitution of cyclohexene rings, thereby producing the pentacyclic morphinan ring system (8).
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 or codeine (3). Morphine accumulation in the plant appears to be related to differentiation of a latex system (12). In efforts aimed at the metabolic engineering of the P. somniferum alkaloid profile as well as at developing alternate biotechnological sources of morphinans, we searched first to identify suitable genetic targets.
We report herein the molecular characterization of salutaridinol 7-O-acetyltransferase of morphine biosynthesis in P. somniferum. The native enzyme was purified to apparent homogeneity, amino acid sequences were determined for internal peptides, and a cDNA clone was generated by RT-PCR 1 using P. somniferum mRNA as template. Heterologous expression in a baculovirus vector in insect cells yielded functional enzyme, and enzymic properties were determined for the recombinant protein. This is the second gene specific to morphine biosynthesis that has been isolated and characterized (13).

EXPERIMENTAL PROCEDURES
Plant Material-Cultured suspension cells of opium poppy P. somniferum were provided by the cell culture laboratories of the Lehrstuhl fü r Pharmazeutische Biologie and the Leibniz-Institut fü r Pflanzenbiochemie. Cultures were routinely grown in 1-liter conical flasks containing 400 ml of Linsmaier-Skoog medium (14) over 7 days at 23°C on a gyratory shaker (100 rpm) in diffuse light (750 lux). Differentiated P. somniferum, Papaver bracteatum, Papaver orientale, Papaver nudicaule, Papaver atlanticum, Papaver rhoeas, and Chelidonium majus plants were grown outdoors in Upper Bavaria or in Saxony-Anhalt, Germany. P. somniferum ssp. setigerum plants were grown in a greenhouse at 24°C with 18 h of light and 50% humidity.
Purification of Native Enzyme and Amino Acid Sequence Analysis-Salutaridinol acetyltransferase was purified from P. somniferum cell suspension cultures exactly according to Lenz and Zenk (8). The purified enzyme preparation was subjected to SDS-polyacrylamide gel electrophoresis to remove traces of impurities, and the Coomassie Brilliant Blue R-250-visualized band representing the acetyltransferase was digested in situ with endoproteinase Lys-C as reported previously (15,16). The peptide mixture was resolved by reversed phase HPLC (column, Merck Lichrospher RP18; 5 m (4 ϫ 125 mm); solvent system, 0.1% trifluoroacetic acid (A) and 0.1% trifluoroacetic acid, 60% acetonitrile (B); gradient, 1% per min; flow rate, 1 ml min Ϫ1 ) with detection at 206 nm. Microsequencing of 10 of the peptides was accomplished on an Applied Biosystems model 470 gas-phase sequencer.
Generation of Partial cDNAs from P. somniferum-Partial cDNAs encoding salutaridinol acetyltransferase from P. somniferum were produced by PCR using cDNA generated by reverse transcription of mRNA isolated from 7-day-old suspension-cultured cells. 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, and the bands of approximately the correct size (537 bp) were isolated and subcloned into pGEM-T Easy (Promega) prior to nucleotide sequence determination. The specific sequences of the oligodeoxynucleotide primers used are given under "Results." Generation of Full-length cDNAs-The sequence information requisite to the generation of a full-length cDNA was derived from the nucleotide sequence of the partial cDNA produced as described under "Results." The complete nucleotide sequence was generated in two steps using one salutaridinol acetyltransferase-specific PCR primer (5Ј-GCC GCA GGC CAA CAA GGG TTG AGG TGG-3Ј for 5Ј-RACE and 5Ј-CCC ATC CTG CAC CAG CTA CTT ATC C-3Ј for 3Ј-RACE) and one RACEspecific primer as specified by the manufacturer. The 5Ј-and 3Ј-RACE-PCR experiments were carried out using a Marathon cDNA amplification kit (CLONTECH). RACE-PCR was performed using the following PCR cycle: 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, and the bands of the expected size (1265 bp for 5Ј-RACE and 917 bp for 3Ј-RACE) were isolated and subcloned into pGEM-T Easy prior to sequencing.
The full-length clone was generated in one piece using the primers 5Ј-CCA TGG CAA CAA TGT ATA GTG CTG CT-3Ј and 5Ј-AGA TCG AAT TCA ATA TCA AAT CAA TTC AAG G-3Ј for PCR with P. somniferum cell suspension culture cDNA as template. The final primers used for cDNA amplification contained recognition sites for the restriction endonucleases NcoI and EcoRI appropriate for subcloning into pFastBac HTa (Life Technologies, Inc.) 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 Schematic biosynthetic pathway leading from salutaridinol to morphine in opium poppy. The 7-hydroxy moiety of salutaridinol is activated by the transfer of an acetyl group from acetyl-CoA (AcCoA), which is catalyzed by salutaridinol 7-O-acetyltransferase. Elimination of acetate to form thebaine is a pH-dependent reaction that can proceed spontaneously. The demethylation of thebaine and codeine are each thought to be catalyzed by cytochrome P-450-dependent enzymes.
agarose gel electrophoresis, and the band of the approximately correct size (1440 bp) 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 NcoI and EcoRI. The recombinant plasmid was transposed into baculovirus DNA in the Escherichia coli strain DH10BAC (Life Technologies, Inc.) and then transfected into Spodoptera frugiperda Sf9 cells according to the manufacturer's instructions. The insect cells were propagated, and the recombinant virus was amplified according to Kutchan et al. (17) and Pauli and Kutchan (18). INSECT-XPRESS serum-free medium (Bio Whittaker) was used in the enzyme expression experiments.
After infection of 150 ml of suspension grown insect cells had proceeded for 3-4 days at 28°C and 130 rpm, the cells were removed by centrifugation under sterile conditions at 1000 ϫ g for 10 min at 4°C. All subsequent steps were performed at 4°C. The pellet was discarded, and the medium was slowly brought to 80% saturation with ammonium sulfate under constant slow stirring. The precipitated proteins were collected by centrifugation at 10,000 ϫ g for 30 min at 4°C. The pellet was dissolved in a minimal volume of 0.5 M NaCl, 10 mM ␤-mercapto-ethanol, 2.5 mM imidazole, 20 mM Tris-HCl adjusted finally to pH 7.0 and was dialyzed for 12-16 h against this same buffer. The His-tagged salutaridinol acetyltransferase was purified by affinity chromatography using a cobalt resin (Talon, CLONTECH) according to the manufacturer's instructions.
General Methods-Latex was collected and resolved as described previously (19,20). Low molecular weight compounds were removed from the supernatant of the resolved latex by passage through a PD 10 column (Amersham Pharmacia Biotech) into 20 mM Tris, 10 mM ␤-mercaptoethanol, pH 7.5. Total RNA was isolated, and RNA gels were run and blotted as described previously (18). Genomic DNA was isolated, and DNA gels were run and blotted according to Decker et al. (21). cDNA clones were labeled by PCR labeling with [␣-32 P]dATP. Hybridized RNA on RNA gel blots and DNA on DNA gel blots were visualized with a STORM PhosphorImager (Molecular Dynamics). The entire nucleotide sequence on both DNA strands of the full-length clone was determined by dideoxy cycle sequencing using internal DNA sequences for the design of deoxyoligonucleotides as sequencing primers. Saturation curves and double reciprocal plots were constructed with the Fig. P program (Version 2.7, Biosoft, Cambridge, United Kingdom). The influence of pH on enzyme activity was monitored in sodium citrate-(pH 4 -6), sodium phosphate-(pH 6 -7.5), Tris-HCl-(pH 7.5-9), glycine/ NaOH-(pH 9 -10.5), and CAPS-buffered (pH 10 -12) solutions.

RESULTS
Purification and Amino Acid Sequence Analysis of Salutaridinol 7-O-Acetyltransferase-Salutaridinol 7-O-acetyltransferase was purified to apparent electrophoretic homogeneity from opium poppy cell suspension cultures, and the amino acid sequence of 10 endoproteinase Lys-C-generated peptides was determined. The sequences and relative positions of these internal peptides are indicated by unshaded boxes in Fig. 2. A comparison of these amino acid sequences with those available in the GenBank TM /EMBL sequence data bases indicated no relevant similarity to known proteins. PCR primer pairs based on a series of salutaridinol 7-O-acetyltransferase peptide combinations also yielded only DNA fragments of irrelevant sequence.
Isolation of the cDNA Encoding Salutaridinol 7-O-Acetyltransferase-During the course of the initial RT-PCR experiments, sequence comparison information appeared in the literature for another acetyltransferase of plant alkaloid biosynthesis (22). The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase was homologous to a series of other putative plant acetyltransferases. A conserved region near the carboxyl terminus of the proteins was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutar-  The positions of these peptides are indicated by arrows in Fig. 2. RT-PCR performed with this primer pair yielded a DNA product of the correct size and sequence for the opium poppy acetyltransferase. RACE-PCR was then used to generate the 5Ј-and 3Ј-portions of the cDNA using nondegenerate nucleotide sequence information provided from the original PCR product.
Sequence Analysis of salAT-Translation of the complete nucleotide sequence of salAT yielded a polypeptide of 474 amino acids containing no apparent signal peptide. This is consistent with the cytosolic localization of the enzyme activity (6). The enzyme activity is also operationally found to be associated with the cytosolic fraction of exuded latex. The salAT amino acid sequence contains residues conserved in other plant acetyltransferases as indicated by the black boxes in Fig. 2. The longest contiguous region of conserved amino acids are the five residues DFGWG near the carboxyl terminus that were used for primer design and are indicated by an arrow. Conserved histidine and aspartate residues (HXXXD, denoted by # in Fig.  2) thought to be involved in catalysis as characterized by x-ray crystallography for the bacterial enzymes chloramphenicol acetyltransferase and dihydrolipoamide acetyltransferase are also present in salutaridinol 7-O-acetyltransferase (23,24). Covalent modification of salutaridinol 7-O-acetyltransferase by treatment with diethylpyrocarbonate (DEPC) resulted in the inhibition of enzyme activity (50% inhibition at 3 mM DEPC and 92% inhibition at 5 mM DEPC) (25). The inactivation by 5 mM DEPC was reduced from 92% to 46% by preincubation of the enzyme with 30 mM acetyl-CoA.
Genomic DNA and Gene Expression Analysis-A genomic DNA gel blot analysis of salAT in P. somniferum is presented in Fig. 3. The restriction endonucleases ApoI, SalI, SpeI, and HincII each recognized one hydrolysis site within the salAT open reading frame yielding two hybridizing bands on the Southern blot. There were no recognition sites for HindIII in the open reading frame. Correspondingly, only a single band hybridized, but it was of approximately one-half the predicted length. This indicates the possible presence of a small intron in the gene. Three recognition sites were present for MspI, theoretically resulting in four hybridizing DNA fragments. Two hybridizing bands of predictable length should have been present, one at 180 bp and one at 511 bp. The absence of these two bands also indicates that intron(s) may be present in the gene. No recognition site was present for EcoRI in the open reading frame, but two hybridizing bands were present on the gel blot also suggesting an intron, which contains an EcoRI restriction site. A more thorough analysis of this point awaits isolation of a genomic clone. These results, taken together, support a single gene hypothesis but do not exclude two very similar, clustered alleles. This is in stark contrast to the other known morphine-specific biosynthetic gene cor1 encoding codeinone reductase for which at least six alleles are expressed (13). RNA gel blot analysis suggests that, as for cor1, salAT is expressed in root, stem, leaf, and capsule of the mature poppy plant (Fig. 4A) (12,13). There appears to be no organ-specific expression of either of these morphine biosynthetic genes. Analysis of RNA from several members of the genus Papaver demonstrated salAT transcript accumulation in 3-week-old seedlings of P. orientale and P. bracteatum although not in P. atlanticum or P. nudicaule (Fig. 4B). P. orientale accumulated the alternate biosynthetic precursor oripavine, and P. bracteatum accumulated the morphine biosynthetic precursor thebaine; both structures contain the oxide bridge formed by the action of salutaridinol 7-O-acetyltransferase. It was, therefore, expected that these two species should contain hybridizing salAT transcript. Nei-ther P. atlanticum nor P. nudicaule contained an alkaloid with the morphinan skeleton, consistent with the absence of transcript in these two species.
Purification and Functional Characterization of Recombinant Enzyme-The salAT cDNA was constructed to express the recombinant protein with six histidine residues elongating the amino terminus. The protein was then purified from S. frugiperda Sf9 cell culture medium in two steps (ammonium sulfate precipitation/dialysis and cobalt affinity chromatography) to yield electrophoretically homogeneous enzyme with an overall yield of 25% and 22-fold purification (Fig. 5). Per liter, the insect cell culture typically produced 2.0 mg (150 nmol s Ϫ1 ) of recombinant enzyme.
Radioassay of pure, recombinant enzyme using [7-3 H]salutaridinol as substrate resulted in 100% conversion into a product that co-migrated during TLC with authentic thebaine standard (Fig. 6A-C). The positive ion electrospray mass spectrum of the enzymic product produced when salutaridinol was used as substrate correlated well with that of thebaine standard (Fig. 6, D and E). The apparent K m value for salutaridinol was determined to be 9 M at a fixed concentration of acetyl-CoA of 30 mM. The apparent K m value for acetyl-CoA was determined to be 54 M at a fixed concentration of salutaridinol of 10 mM. The V max for the acetylation of salutaridinol was 25 pmol s Ϫ1 with a temperature optimum of 47°C and a pH optimal range of 7-9 under standard assay conditions. The recombinant enzyme acetylated 7(S)-salutaridinol and nudaurine (apparent K m for nudaurine, 23 M at 30 mM acetyl-CoA; apparent K m for acetyl-CoA, 106 M at 10 mM nudaurine; V max , 19 pmol s Ϫ1 ) at C-7 but not 7(R)-salutaridinol, salutaridine, codeine, morphine, or deacetylvindoline. The kinetic values and chemical structures for the alkaloidal substrates salutaridinol and nudaurine are summarized in Table I. As designated by the ratio k cat /K m (salutaridinol):k cat /K m (nudaurine), the enzyme acetylated salutaridinol preferentially to nudaurine by a factor of 3.3. DISCUSSION We have isolated a full-length cDNA clone that encodes salutaridinol 7-O-acetyltransferase from cell suspension culture of opium poppy P. somniferum. An identical match was observed between the deduced and directly determined amino acid sequences of ten internal peptides distributed throughout the open reading frame. The identity of the clone was ascertained by catalytic activity of the functionally expressed recombinant enzyme. The results obtained by RACE-PCR indicated that the reading frame is 1425 nucleotides long corresponding to 474 amino acids. These values correlate well to the transcript size obtained by RNA gel blot analysis. The calculated molecular mass of the enzyme is 52.6 kDa, which is consistent with the apparent molecular mass of 50 kDa determined by SDS-polyacrylamide gel electrophoresis (8).
Acyltransferases of related amino acid sequence develop into a family of genes with integral roles in plant secondary metabolism. This has been suggested previously by De Luca (22). The amino acid sequence of salutaridinol 7-O-acetyltransferase is similar to acyltransferases involved in monoterpenoid indole alkaloid, phenylpropanoid conjugate, and diterpenoid formation (22, 26 -29). Histidine and aspartate residues ( 163 HXXXD 167 ) are highly conserved as is a DFGWG motif near the carboxyl terminus of the proteins. The equivalent histidine residue has been shown through site-directed mutagenesis or chemical modification to be essential for catalytic activity in other acyltransferases (30). Carbethoxylation of histidine residues in salutaridinol 7-Oacetyltransferase with DEPC resulted in a loss of enzyme activity. Preincubation of the enzyme with acetyl-CoA partially protected a putative active site histidine residue from chemical The O-Acetyltransferase of Morphine Biosynthesis modification and resultant inactivation. A catalytic triad (Ser-His-Asp) as found in serine proteases and lipases has been postulated for other acyltransferases (30). The crystal structure of arylamine N-acetyltransferase from Salmonella typhimurium indicates that a cysteine residue may be a component of the catalytic triad (Cys 69 , His 107 , Asp 122 ) (31). The amino acid sequence of salutaridinol 7-O-acetyltransferase contains both a conserved serine (Ser 33 ) and a conserved cysteine (Cys 152 ) suggesting that a catalytic triad could also be essential to enzyme activity in this family of plant acyltransferases. This consensus information should certainly also aid in the identification and isolation of additional members of this family that may be involved in other plant secondary pathways. The site of morphine biosynthesis in P. somniferum is a long-standing question. We examined herein the expression pattern of salAT in P. somniferum. salAT was expressed in each major plant part analyzed-root, stem, leaf, and capsule. This corresponds to the detection of transcript of another morphine biosynthesis-specific gene, cor1, in each plant organ analyzed (12). Additionally, salutaridinol 7-O-acetyltransferase and codeinone reductase enzyme activity have each been detected in the cytosolic fraction of isolated latex (12,13). The gene cyp80b1 participates in (S)-reticuline biosynthesis occurring before a bifurcation in the biosynthetic pathway that leads to more than 80 isoquinoline alkaloids. cyp80b1 is, therefore, common to several biosynthetic pathways including morphine, sanguinarine, and noscapine. Transcript of cyp80b1 was also detected in all plant organs analyzed (12). Accumulation of morphinan alkaloids is thought to correlate with the appearance of laticifer cells in the developing plant and in differentiating plant cell culture (32,33). A reticulated laticifer system associated with the vascular tissue is present through the aerial parts of the poppy plant. In roots, nonreticulated laticifers are present (34,35). The localization of three genes of morphine biosynthesis, cyp80b1, salAT, and cor1, is thus far consistent with the assumption this biosynthesis is, at least in part, associated with laticifer cells. Interestingly, deacetylvindoline acetyltransferase has been localized to laticifer cells in aerial parts of C. roseus (36).
With even more morphine biosynthetic genes now in hand, we can begin to address the question of why only P. somniferum produces morphine, whereas other Papaver species such as P. rhoeas, P. orientale, P. bracteatum, P. nudicaule, and P. atlanticum do not. salAT transcript was detected in RNA isolated from P. somniferum, P. orientale, and P. bracteatum but not in RNA from P. nudicaule and P. atlanticum. This is consistent with the expected distribution based upon accumulation of alkaloids having the morphinan nucleus in these species (i.e. morphine in P. somniferum, thebaine in P. bracteatum, and oripavine in P. orientale). This is in sharp contrast to those results obtained for cor1 transcript, which was detected also in Papaver species that are not known to accumulate codeine (12). The genes of alkaloid biosynthesis in P. somniferum will certainly continue to provide useful information on the molecular evolution of plant secondary metabolism in latex systems.
An important aspect of our molecular genetics experiments with P. somniferum is an eventual biotechnological production of thebaine, codeine, and morphine. We have isolated cDNAs encoding several enzymes of morphine biosynthesis. The first enzyme in the biosynthetic pathway for which we have isolated a cDNA is norcoclaurine 6-O-methyltransferase (37). The second enzyme is the cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3Ј-hydroxylase (12,18). These enzymes are common to the morphine, noscapine, and sanguinarine biosynthetic pathways. Specific to morphine biosynthesis are salutar-idinol 7-O-acetyltransferase (reported herein) and codeinone reductase, the penultimate enzyme of the morphine pathway that reduces codeinone to codeine (13). A cDNA encoding an enzyme involved generally in metabolism but essential to the activity of the cytochrome P-450-dependent monooxygenase, the cytochrome P-450 reductase has also been isolated (38). Each of the cDNAs has been functionally expressed in insect cell culture (S. frugiperda Sf9 cells) or in E. coli. An immediate application of these cDNAs is in the metabolic engineering of P. somniferum to obtain altered alkaloid profiles in the plant. A more long term goal is a biomimetic synthesis of morphinan alkaloids combining chemically and enzymatically catalyzed steps. For this latter application, additional cDNAs encoding enzymes that mediate transformations occurring between (R)-reticuline and morphine still need to be isolated.