Cytochrome P450 (P450) 2 enzymes catalyze the most versatile chemical reactions in nature (1). There is, however, a discrepancy between the plant and the animal kingdoms with regard to the sheer number of these biocatalysts. Whereas in a single model plant, Arabidopsis thaliana, there are to date 273 P450 proteins, in the human genome, only 57 of these proteins are present. Whereas plants and animals share a multitude of highly regio-and stereospecific O-demethylation reactions, more complex reactions such as phenol coupling are much more abundant in plants than in animals, especially in the alkaloid field (2-11). The proposal of Barton and Cohen (12) correlated the structure of specific plant alkaloids in terms of this reaction mechanism and gave mechanistic proposals of how these phenol-coupled products may possibly be biosynthesized in nature. The oxidation of phenols by one-electron transfer affords radicals, which, by radical pairing, form new C-C or C-O bonds either by intra-or intermolecular coupling. The first two examples that unequivocally demonstrated the formation of C-C and C-O bonds in a stereo-and regioselective manner in plant metabolism are catalyzed by specific P450-linked microsomal-bound plant enzymes (13). One of these enzymes was salutaridine synthase from Papaver somniferum (opium poppy) (14). This synthase catalyzes the intramolecular formation of the critical C12-C13 carbon bridge and is a key enzyme in morphine biosynthesis.The groups of Goldstein (15) and Spector (16) have published a number of reports over the past 25 years claiming that mammals are capable of synthesizing de novo traces of endogenous morphine. However, no convincing experimental data have been presented regarding the enzymes. Phenol-coupling reactions in mammals are extremely rare, and the only example described thus far is the formation of thyroxine, by radical pairing, in humans. If the key step of morphine synthesis, the formation of phenol-coupled salutaridine from (R)-reticuline, occurs in mammals then in analogy to plants, a P450 enzyme must be present (in mammals) to catalyze this reaction. In 1987, the first experiments were conducted in an attempt to discover the reaction by supplying uniformly labeled racemic [ 3 H]reticuline in the presence of rat microsomes and NADPH to examine whether [ 3 H]salutaridine can be formed under these conditions (15). A radioactive compound was formed in 1% yield and assumed to be the phenol-coupled product salutaridine. We later repeated this experiment using (R)-[N-14 CH 3 ]reticuline and NADPH-fortified microsomes from pig, rat, cow, and sheep. We observed the formation of [N-14 CH 3 ]salutaridine with the correct stereochemistry at carbon 9 (17). The enzyme from porcine liver was subsequently purified to homogeneity and the reaction product was characterized by mass spectrometry and physical parameters to be a product of a P450 enzyme, which we provisionally named "salutaridine synthase" (18). The aim of this report is the identification of the homogenous porcine P450 e...
Along the poppy morphine biosynthetic pathway, the transition from salutaridinol-7-O-acetate to thebaine had previously been claimed to proceed non-enzymatically between pH 8-9. At pH 6-7, the acetate was transformed to an azonine derivative. These transformation reactions were revisited using Papaver somniferum protein extracts in search for a possible protein catalyst. After removal of residues and inhibitory low molecular weight compounds from the latex of P. somniferum, this latex serum was shown to convert salutaridinol-7-O-acetate to thebaine in high yield at the physiological pH of 7.0. A new enzyme, which was partly purified, was shown to catalyze this reaction and was partly characterized. As a result, a further enzymatic step had to be added to the morphine pathway in the poppy plant, involved in the transformation of 2 moles L-tyrosine to one mole of morphine.
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