Morphine biosynthetic pathway

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... 

The first steps of the morphine biosynthetic pathway, outlined in Scheme 1,... 

A minor alternative route in the morphine biosynthetic pathway involves the demethylation of the 3-methyl group of thebaine by CODM to generate oripavine. The latter is demethylated by T60DM to morphinone, which is ultimately reduced by COR to morphine (Figure 6.20) [102]. 

Genomic and transcriptomic technologies have been used to rapidly identify biosynthetic steps. There are currently over 40,000 expressed enzyme tags (ESTs) generated fi om alkaloid-producing plants that have been used to isolate genes involved in the alkaloid pathway [7]. Some alkaloid biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes, for example, conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may catalyze two or more separate reactions in the pathway, for example, hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Alkaloid biosynthesis also involves compartmentalization. Tissue-specific localization studies have shown that sequential biosynthetic enzymes can occur in distinct cell types [8, 9]. During the biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus, different enzymatic steps are carried out in different cellular compartments (Fig. 8.5) [10]. Various steps in the pathway are carried out in different types of cell. This requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis also involves two different cell types. 

Mishra S, Meena A, Singh S, Yadav DK, Khan F, Shukla RK (2010) Detection of substrate binding motifs for morphine biosynthetic pathway intermediates in novel wound inducible (R,S)-reticuline 7-O-methyltransferase of Papaver somniferum. In Proceedings of international symposium on current status and opportunities in Aromatic Medicinal Plants (AROMED), CIMAP (CSIR), Lucknow, India, 21-24 Feb 2010, Session I P-25, p 51... 

Morphine biosynthetic pathway, 428 Morphine-3-glucuronide (M-3-G), 1376 Morphine-6-glucuronide (M-6-G), 441, 1376 Morroniside (isolated as 7a/7Pmixture), 3028 MPTP. See 1-Methy 1-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)... 

At the subcellular level, the morphine biosynthetic pathway combines several cytochrome P-450 enzymes, bound to ER membranes (Chou and Kutchan, 1998) with soluble enzymes that reside in ER derived vesicles, e.g. norcoclaurine synthase, or in the cytosol, e.g. codeinone reductase (Zenk, 1994 Facchini and St-Pierre, 2005). The diverse localization of the biosynthetic enzymes requires several transport steps of the intermediates between cytosol, vesicles and vacuoles. Only one of them has been characterized by pioneering experiments (Deus-Neumann and Zenk, 1986) vacuolar vesicles prepared from Fumaria capreolata accumulate (S)-reticulin or (S)-scoulerin via highly specific transporters, that discriminate between (S)- and (R) stereoisomer and exclude other benzylisoquinolines (sanguinarine, protopine, morphine) and alkaloids of unrelated families (e.g. indoles or tropanes). Uptake is energized by the pH gradient across the tonoplast. Accumulated... 

With a morphine biosynthetic gene in hand, we believed we could begin to address the question why only P. somniferum produces morphine, while other Papaver species such as P. rhoeas, P. orientale, and P. bracteatum do not. Unexpectedly, we found that the codeinone reductase transcript was present to some degree in all four species investigated. A review of the literature revealed no alkaloids reported in P. rhoeas for which codeinone reductase should participate in the synthesis. Similarly, P. orientale accumulates the alternate morphine biosynthetic precursor oripavine, but codeinone reductase is not involved in the biosynthesis of oripavine, acting instead after this alkaloid along the biosynthetic pathway to morphine.22 P. bracteatum produces the morphine precursor thebaine as a major alkaloid. As for oripavine in P. orientale, codeinone reductase would act in P. bracteatum after thebaine formation on the pathway to morphine. It appears, therefore, that the reason that P. rhoeas, P. orientale, and P. bracteatum do not produce morphine is not related to the absence of the transcript of the morphine biosynthesis-specific gene codeinone reductase. The expression of codeinone reductase may simply be an evolutionary remnant in these species. 

Scheme 30 The overall biosynthetic pathway from tyrosine to morphine...

Alkaloid biosynthetic pathways are under strict regulation in plants. Until now, our limited knowledge of the fundamental mechanisms involved in the control of alkaloid metabolism has severely restricted our ability to harness the vast biotechnological potential of these important secondary pathways. For example, the use of plant cell cultures for the commercial production of pharmaceutical alkaloids has not become a reality despite decades of empirical research. The application of traditional and modem biochemical, molecular, and cellular techniques has revealed important clues about the reasons why C. rosens cultures accumulate tabersonine and catharanthine, but not vindoline or vinblastine, and why opium poppy cultures produce sanguinarine, but not codeine or morphine. The inability of dedifferentiated cells to accumulate certain metabolites was interpreted as evidence that the operation of many alkaloid pathways is tightly coupled to the development of specific tissues. Recent studies have shown that alkaloid pathways are regulated at multiple levels,... 

Some of the most interesting applications of organic structural theory to the elucidation of biosynthetic pathways were stimulated by efforts to formulate mechanisms for the biosynthesis of alkaloids. Conversely, consideration of implied biogenetic relations have occasionally helped structural determination. An important aspect of theories concerning alkaloid biosynthesis has been the assumed role of the aromatic amino acids in their formation. Only limited experimental evidence is available in this area. The incorporation of tyrosine- 8-C into morphine has been shown to be in accordance with a theory for its formation from 3,4-dihydroxyphenyl-alanine plus 3,4-dihydroxyphenylacetaldehyde. A stimulating theory of the biosynthesis of indole alkaloids, based on a condensation between trypt-amine and a rearrangement product of prephenic acid, has recently been published. The unique stereochemistry of C15 of these alkaloids had an important part in the formulation of the theory. Experimental proof of this theory would be valuable for several areas of alkaloid chemistry and biosynthesis. 

Figure 4 Redox reactions in biosynthetic pathways. A one-electron oxidation of two coniferyi aicohoi monomers is the initiating step in podophyllotoxin biosynthesis, and a simiiar one-eiectron oxidation piays a key role in the biosynthesis of the aikaioid morphine. Redox reactions are also used to crosslink nascent scaffoids, as shown for vancomycin, or to add oxygen-based functionality to reduced scaffoids, as shown for taxoi.

There are two possible biosynthetic pathways for the conversion of thebaine to morphine, Fig. (67) [148]. One is orthodoxically known in the litreatures [4, 130, 131], that is morphine biosynthesis from thebaine via codeinone and codeine. Another one i.e. the first ever demonstrated by Brochmann-Hanssen in 1984 [149], is the biosynthesis via oripavine and morphinone. The transformed clone could synthesize codeine but lacked morphine though the non-transformed clone obtained from the same plant material accumulated morphine at the latter developmental stage (Table 21). This suppressed morphine content was also observed in the opium derived from the transformed P. somniferum plants that had been... 

Fig. (67). Two possible biosynthetic pathways for the conversion of thebaine to morphine.

P. somniferum) in this biosynthetic pathway is still obscure. InP. somni-ferum the biosynthesis of morphine can also proceed from (+ )-reticuline provided that this compound is first transformed into (— )-reticuline. The biosynthesis is, however, impossible (via thebaine) from ( + )-reticuline methochloride (52). 

The biosynthetic pathway which is indicated by the above results is illustrated in Scheme 11 norlaudanosoline (70) had previously been shown to be a morphine (71) precursor. The implication of a keto-acid rather than an aldehyde in benzyliso-quinoline formation accords with observations on the biosynthesis of simpler isoquinolines and stands in contrast with the utilization of an aldehyde, sec-ologanin, in a similar reaction in the biosynthesis of terpenoid indole alkaloids. ... 

Figure 12.13. The biosynthetic pathway for L-tyrosine derived tetrahydrobenzylisoquinoline alkaloids. The core production of (5)-reticuline and the branch points for berberine, macarpine, and morphine production. Reprinted with permission from Chou and Kutchan (I998)" .

The very short and highly efficient biomimetic synthesis of morphine [16, 32] by Rice stands out in terms of overall yield and brevity no subsequent contribution to this area exceeds this milestone achievement. The route follows the biosynthetic pathway and delivers dihydrocodeinone in almost 30% overall yield. 

Other constraints are important for more complex products, for which mass is not central, but value. For example, central nervous system stimulants are a new class of substances addressed by production with engineered baker s yeast. Expressing the biosynthetic pathways for the opioids thebaine and hydrocodone, and parts of the morphine pathway in yeast, a first step is taken for easy production of opiates [21, 22]. This opens the possibility for the development of new painldllers with less addictive potential. However, it clearly is a new technology that could be abused with many negative consequences - so some contemplation about how to control these developments seems advisable [23]. Not only are narcotics in the center of interest but stimulants such as caffeine and theobromine have also recently been produced with genetically engineered 5. cerevisiae strains [24]. 

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