morphinans and norreticuline

The biosynthesis of morphine, a stereochemically complex alkaloid, has been shown to occur in plants and animals. A search in the human genome for methyltransferases capable of catalyzing the N-methylation of benzylisoquinoline alkaloids, as biosynthetic precursors of morphine, yielded two enzymes, PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49). Introduction of an N-terminal poly-histidine tag enabled purification of both proteins by immobilized metal affinity chromatography. Recombinant PNMT and NMT were characterized for their catalytic activity towards four benzylisoquinolines: tetrahydropapaveroline (THP), 6-O-methyl-THP, 4'-O-methyl-THP and norreticuline. Human PNMT accepted none of the offered alkaloids and was only active with its established substrate, phenylethanolamine. The second enzyme, human NMT, converted all four benzylisoquinolines, however, with a strict preference for (R)-configured morphine precursors. Determination of kinetic parameters of NMT for the four (R)-configured benzylisoquinoline alkaloids by LC-MS/MS revealed (R)-norreticuline to be the best substrate with an even higher catalytic activity as compared to the previously reported natural substrate tryptamine. In addition, isolation of the morphine precursor salutaridine from urine of mice injected (i.p.) with (R)-THP provides new evidence that the initial steps of morphine biosynthesis in mammals occur stereochemically and sequentially differently than in plants and suggests an involvement of the herein characterized (R)-specific NMT.

Topics: Alkaloids; Animals; Base Sequence; Benzylisoquinolines; DNA Primers; Ethanolamines; Humans; Isoquinolines; Kinetics; Methyltransferases; Mice; Morphinans; Morphine; Phenylethanolamine N-Methyltransferase; Recombinant Proteins; Stereoisomerism; Substrate Specificity

A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.

Topics: Alkaloids; Benzylisoquinolines; Centrifugation, Density Gradient; Enzyme Stability; Isomerism; Isoquinolines; Mass Spectrometry; Morphinans; Oxidoreductases; Papaver; Seedlings; Vacuoles