Phenylpropanstoffwechsel

Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

Two new amide-linked conjugates of jasmonic acid, N-[(3R,7R)-(−)-jasmonoyl]-(S)-dopa (3) and N-[(3R,7R)-(−)-jasmonoyl]-dopamine (5), were isolated in addition to the known compound N-[(3R,7R)-(−)-jasmonoyl]-(S)-tyrosine (2) from the methanolic extract of flowers of broad bean (Vicia faba). Their structures were proposed on the basis of spectroscopic data (LC-MS/MS) and chromatographic properties on reversed and chiral phases and confirmed by partial syntheses. Furthermore, tyrosine conjugates of two cucurbic acid isomers (7, 8) were detected and characterized by LC-MS. Crude enzyme preparations from flowers of V. faba hydroxylated both (±)-2 and N-[(3R,7R/3S,7S)-(−)-jasmonoyl]tyramine [(±)-4] to (±)-3 and (±)-5, respectively, suggesting a possible biosynthetic relationship. In addition, a commercial tyrosinase (mushroom) and a tyrosinase-containing extract from hairy roots of red beet exhibited the same catalytic properties, but with different substrate specificities. The conjugates (±)-2, (±)-3, (±)-4, and (±)-5 exhibited in a bioassay low activity to elicit alkaloid formation in comparison to free (±)-jasmonic acid [(±)-1].

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.