Acetyl Coenzyme A:Salutaridinol-7-O-Acetyltransferase from Papaver somniferum Plant Cell Cultures:

Abstract

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.

Molecular Characterization of the Salutaridinol 7-O-Acetyltransferase Involved in Morphine Biosynthesis in Opium Poppy Papaver somniferum

“…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).…”

“…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).…”

“…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).…”

How human neuroblastoma cells make morphine

“…Cell-free preparations of rat liver catalyzed the acetylation of salutaridinol but not 7-episalutaridinol (data not shown). In light of these findings, it is very likely that the formation of thebaine from salutaridinol follows the same mechanism in human cells that has been proven for opium poppy (30).…”

“…However, morphine obtained from control SH-SY5Y cells (grown under standard conditions without isotopelabeled compound) showed not only the [M] •ϩ peak at m͞z 429 [M] •ϩ but also the naturally occurring 13 Ring closure to form the ether linkage during the transformation of salutaridinol to thebaine is the result of a nucleophilic attack of the phenol group on the dienol system and subsequent displacement of the hydroxyl (S N 2Ј-elimination). In Papaver plants, an additional reaction is used to improve the nature of the leaving group, which is accomplished by means of acetylation by a highly substrate-specific and stereospecific enzyme, acetyl-CoA: salutaridinol-7-O-acetyltransferase (30). The subsequent ring closure seems to occur without enzyme participation at pH values between 8 and 9 in the cytosol of the plant cell.…”