Salutaridinol 7 0 acetyltransferase

The localization of isoquinoline biosynthesis has been investigated at the cellular level in intact poppy plants by using in situ RNA hybridization and immunoflouresence microscopy. The localization of 4 -OMT (reticuline biosynthesis), berberine bridge enzyme (saguinarine biosynthesis), salutaridinol acetyltransferase (morphine biosynthesis), and codeinone reductase (morphine biosynthesis) has been probed. 4 -OMT and salutaridinol acetyltransferase are localized to parenchyma cells, whereas codeinone reductase is localized to laticifer cells in... 

Salutaridinol 7-0-acetyltransferase catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-Oacetate, the immediate precursor of thebaine along the morphine biosynthetic pathway in P. somniferum (Fig. 10.7).26 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 (reviewed in2). An acetyl CoA-dependent acetyltransferase also participates in vindoline biosynthesis in Catharanthus roseus, the source of the chemotherapeutic dimeric indole alkaloid vinblastine (reviewed in2). Acetyl CoA deacetylvindoline 4-O-acetyltransferase catalyzes the last step in vindoline biosynthesis. A cDNA encoding acetyl CoA deacetylvindoline 4-0-acetyltransferase was recently successfully isolated.27... 

Salutaridinol 7-O-acetyltransferase was purified to apparent electrophoretic homogeneity from P. somniferum cell suspension cultures and the amino acid sequence of ten endoproteinase Lys-C-generated peptides was determined.28 A comparison of these amino acid sequences with those available in the GenBank/EMBL sequence databases indicated no relevant similarity to known proteins. The first attempt to isolate a cDNA encoding salutaridinol 1-0-... 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

RNA gel blot analysis of several members of the genus Papaver demonstrated that salutaridinol 7-O-acetyltransferase transcript accumulated in three-week-old seedlings of P. orientale and P. bracteatum, though not in P. atlanticum or P. nudicaule % P. orientale accumulates the alternate biosynthetic precursor oripavine, and P. bracteatum accumulates the morphine biosynthetic precursor... 

Fig. 10.8 Selected cDNAs isolated in recent years that encode enzymes involved in the biosynthesis of various classes of isoquinoline alkaloids. 6-OMT, norcoclaurine 6-0-methyltransferase 23 CYP80A1, berbamunine synthase 19 CYP80B1, (S)-A-methylcoclaurine 3 -hydroxylase 20 CPR, cytochrome P-450 reductase 29 4 -OMT, (5)-3 -hydroxy-A-methylcoclaurine 4 -0-methyltransferase 30 BBE, berberine bridge enzyme 12 SalAT, salutaridinol 7-O-acetyltransferase 28 COR, codeinone reductase.25...

LENZ, R., ZENK, M.H., Acetyl coenzyme A salutaridinol 7-O-acetyltransferase from Papaver somniferum plant cell cultures, J. Biol. Chem., 1995, 270, 31091-31096. 

GROTHE, T., LENZ, R KUTCHAN, T.M., Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum, J. Biol. Chem. 2001,276, 30717-30723. 

The isolation of the cDNAs encoding the enzymes involved in diverse isoquinoline alkaloid formation in plants and microorganisms allowed the first metabolic engineering routes to be developed and paved the way for new ways of future production of isoquinoline alkaloids. For instance, transgenic opium poppy plants were created in which codeinone reductase was suppressed by RNAi, resulting in the substitution of morphine synthesis with the non-narcotic precursor reticuline [110]. In a similar approach, RNAi suppression or overexpression of salutaridinol 1-0-acetyltransferase in opium poppy led to accumulation of salutaridine or increase of morphine, codeine and thebaine content [111], suppression of the BBE led to accumulation of berberine in California poppy cells [112],... 

Conversion of (S)-reticuline to its ( )-epimer is the first committed step in morphinan alkaloid biosynthesis in certain species. 1,2-Dehydroreticuline reductase catalyzes the stereospecific reduction of 1,2-dehydroreticuline to (7 )-reticuline.39 Intramolecular carbon-carbon phenol coupling of (if)-reticuline by the P450-dependent enzyme salutaridine synthase (STS) results in the formation of salutaridine.40 The cytosolic enzyme, salutaridine NADPH 7-oxidoreductase (SOR), found in Papaver bracteatum and P. somniferum, reduces salutaridine to (7S)-salutaridinol.41 Conversion of (7S)-salutaridinol into thebaine requires closure of an oxide bridge between C-4 and C-5 by acetyl coenzyme A salutaridinol-7-0-acetyltransferase (SAT). The enzyme was purified from opium poppy cultures and the corresponding gene recently isolated (Fig.7.2).42,43 In the last steps of morphine... 

In the next step, acetylation of the hydroxyl at Cl utilizing acetyl-CoA (acetyl-CoA is the cofactor that is used by the enzyme salutaridinol 7-O-acetyltransferase [EC 2.3.1.150]) and CoA-SH and 7-O-acetylsalutaridinol results. Spontaneous cyclization accompanies loss of acetic acid, and thebaine results. 

Allen RS et al (2008) Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppressirai of salutaridinol 7-0-acetyltransferase in opium poppy. Plant Biotechnol J... 

Enzyme 2.3.1.150 Salutaridinol 7-0-acetyltransferase class transferases acyltransferases... 

Fig. 4 Pathways of benzylisoquinoline biosynthesis. A selection of biosynthetic enzymes is notified with their localization in the cytosol (yellow), the ER membrane (red) or in the lumen of cytosolic vesicles (blue). Informations are taken mainly from Facchini and St-Pierre (2005), Bock et al. (2002), own experiments and other references cited in the text. BBE, berberine bridge enzyme CFS, cheilanthifoline synthase CNMT, coclaurine N-methyltransferase COR, codeinone reductase DBOX, dihydrobenzophen-anthridine oxidase MSH, N-methylstylopine 14-hydroxylase NCS, norcoclaurine synthase NMCH, N-methylcoclaurme 3 -hydroxylase 4 OMT, 3 -hydroxy-N-methylcoclaurine 4 -0-methyltransferase 60MT, norcoclaurine 6-O-methyltransferase 70MT, reticuline 7-O-methyltransferase P6H, protopine 6-hydroxylase SAT, salutaridinol-7-O-acetyltransferase SOR, salutaridineiNADPH 7-oxidoreductase STS, stylopine synthase SAS, salutaridine synthase TNMT, tetrahydroprotoberberine cis-N-methyltrans-ferase TYDC, tyrosine decarboxylase CAS, canadine sjmthase SOMT, scoulerine 9-O-methyltransferase STOX, (S)-tetrahydroprotoberberine oxidase...

Figure 4.2 Biosynthesis of benzylisoquinoline alkaloids. Enzyme abbreviations TYDC, tyrosine/dopa decarboxylase NCS, norcoclaurine synth lse 60MT, norcoclaurine 6-O-methyltransferase 4 OMT, 3 -hydroxy-/V-methylcoclaurine 4 -0-methyltransferase CYP80A, berbamunine synth lse CYP80B, N-methylcoclaurine 3 -hydroxylase BBE, berberine bridge enzyme SOMT, scoulerine 9-0-methyltransferase CYP719A, (S)-canadine synthase SAT, salutaridinol 7-O-acetyltransferase COR, codeinone reductase.

---