Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Allosteric interaction of the neuromuscular blockers vecuronium and pancuronium with recombinant human muscarinic M2 receptors.

Neuromuscular blocking drugs produce muscle weakness by interaction with nicotinic-acetylcholine receptors. Cardiovascular side effects have been reported. In this study the neuromuscular blocking drug vecuronium and the controls gallamine and pancuronium slowed the rate of atropine induced [(3)H]N-methylscopolamine dissociation from Chinese hamster ovary cells expressing recombinant human muscarinic M2 receptors K(off) values min(-1); vecuronium (125 nM), atropine 0.45+/-0.07+blocker 0.04+/-0.02; gallamine (21 nM), atropine 0.42+/-0.05+blocker 0.15+/-0.04; pancuronium(21 nM), atropine 0.36+/-0.03+blocker 0.03+/-0.01). These data indicate that vecuronium, gallamine and pancuronium interact with an allosteric site on the muscarinic M2 receptor (located on the heart) and this may explain some of their cardiac side effects.

(R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy.

S-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)-reticuline (4.2 sec(-1) mm(-1)), (S)-reticuline (4.5 sec(-1) mm(-1)), (R)-protosinomenine (1.7 sec(-1) mm(-1)), and (R,S)-isoorientaline (1.4 sec(-1) mm(-1)) as well as guaiacol (5.9 sec(-1) mm(-1)) and isovanillic acid (1.2 sec(-1) mm(-1)) are O-methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)-norcoclaurine (7.4 sec(-1) mm(-1)), (R)-norprotosinomenine (4.1 sec(-1) mm(-1)), (S)-norprotosinomenine (4.0 sec(-1) mm(-1)) and (R,S)-isoorientaline (1.0 sec(-1) mm(-1)). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.