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  • 1
    ISSN: 1432-2048
    Keywords: Datura (root, alkaloid) ; Root culture (alkaloid production) ; Scopolamine synthesis ; Tropane alkaloid synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ℴ flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.
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  • 2
    ISSN: 1432-2048
    Keywords: Datura (root, alkaloid) ; Hyoscyamine ; Putrescine ; α-Difluoromethylarginine ; α-Difluoromethy-lornithine ; Arginine decarboxylase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl-α-difluoromethylarginine or dl-α-difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl-α-difluoromethylornithine.
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  • 3
    ISSN: 1432-2048
    Keywords: Arginine decarboxylase ; Datura (root, alkaloid) ; Root culture (alkaloid production) ; Hyoscyamine synthesis ; Tropane alkaloid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.
    Type of Medium: Electronic Resource
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