SummaryThe narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli. Heterologously expressed codeinone reductase-calmodulin-binding peptide fusion protein was puri®ed from E. coli using calmodulin af®nity column chromatography in a yield of 10 mg enzyme l -1 . These four isoforms demonstrated very similar physical properties and substrate speci®city. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6¢-deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6¢-deoxychalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possibly arise from primary metabolism.

Salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) 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 M and 54 M 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.

Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase, a highly substrate-specific enzyme, has been purified nearly 3,000-fold to homogeneity from Papaver somniferum plant cell suspension cultures. Purification was achieved by fractionated ammonium sulfate precipitation, dye-ligand affinity chromatography on matrex red A, gel filtration, ion exchange chromatography on Mono Q and a second dye-ligand affinity chromatography on fractogel TSK AF Blue. The purified enzyme was a single polypeptide with an M r ‫؍‬ 50,000 displaying an isoelectric point of 4.8, a pH optimum between pH 6 and 9 and a temperature optimum at 47°C. The K m values for the substrate salutaridinol and the co-substrate acetyl coenzyme A were 7 and 46 M, respectively. Salutaridinol-7-O-acetyltransferase catalyzes the stoichiometric transfer of the acetyl group from acetyl coenzyme A to the 7-OH group of salutaridinol yielding salutaridinol-7-O-acetate, which is a new intermediate in morphine biosynthesis. Salutaridinol-7-O-acetate undergoes a subsequent spontaneous allylic elimination at pH 8 -9, leading to the formation of thebaine (1), the first morphinan alkaloid with the complete pentacyclic ring system, or at pH 7 leading to dibenz[d,f]azonine alkaloids that contain a nine-membered ring. Acetylation and subsequent allylic elimination is a new enzymic mechanism in alkaloid biosynthesis, which in the poppy plant can transform one precursor into alkaloids possessing markedly different ring systems, depending on the reaction pH.

Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (-)codeinone to (-)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver sonziziferuin L. cell cultures. The purification involved ammonium sulfate precipitation (40-80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 5 5 S ) , affinity chromatography (fractogel TSK AF Blue), ion-exchange chromatography (DEAE-Sephacel) and native PAGE. The purified codeinone reductase was found to be a monomeric protein of 35 i-1 kDa that is highly substratespecific, reducing only the C6 0x0 group of codeinone and morphinone as well as a few analogues. The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1. The temperature optimum is at 40°C and the isoelectric point (pl) at 4.4. The apparent K,,, values (forward reaction) for codeinone and NADPH are 23 pM and 168 pM, respectively. Using capsule tissue of differentiated F! soinn$erum plants as an enzyme source, two codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity. These isoenzymes could not be separated for characterization and showed slightly different kinetic features (K,,, values: codeinone 9 pM; NADPH 81 pM) compared with the cell culture enzyme.

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