Tropane alkaloids biosynthetic pathways

F. (1). Tropane alkaloids biosynthetic pathway. The known enzymes are indicated. ODC (ornithine decarboxylase), ADC (arginine decarboxylase), PMT (putrescine methyl transferase), MPO (methyl putrescine oxidase), TRI, TRII (tropinone reductase I, II), H6H (hyoscyamine 6p hydroxylase). 

In this section, an overview of the known enz5mies participating in the different steps of the tropane alkaloid biosynthetic pathway, as well as the genetic manipulation of these enzymes and the efforts for... 

A-methylputrescine oxidase catalyzes the formation of N-methylpyrrolinium cation, an important intermediate of tropane alkaloids biosynthetic pathway as well as of nicotine. Fig. (1). The... 

The engineering of tropane alkaloids biosynthetic pathway by overexpression genes participating in that pathway constitutes and important tool for scopolamine and hyoscyamine enhancement. Thus, no much attention has been given to TRI and TRII genes and only two reports describe the manipulation of these genes. 

Hyoscyamine 6P-hydroxylase (H6H, EC 1.14.11.11) is a 2-oxoglutarate-dependent dioxygenase and is the final enzyme of the tropane alkaloid biosynthetic pathway. The H6H catalyzes the conversion of hyoscyamine into 6P-hydoxyhyoscyamine and scopolamine by two sequential reactions [24, 25]. Firstly, it catalyses the 6P-hydroxylation of hyoscyamine and secondly, the formation of the epoxy group leading to scopolamine [13]. Several works have revealed that the epoxidation activity is much lower than the hydroxylation one [11, 26]. These findings are in agreement to those reported in our laboratory about the H6H enzyme isolated from Brugmansia Candida (syn. Datura Candida) [27-29]. This South America n native plant is a natural hybrid between B. aurea and B. versicolor and a tropane alkaloids producer [30,31]. 

Robins, R. J., Walton, N. J., Parr, A. J., Aird, E. H., Rhodes, J. C., and Hamill, J. D. (1994) Progress in the genetic engineering of the pyridine and tropane alkaloid biosynthetic pathways of Solanaceous plants. In Genetic Engineering of Plant Secondary Metabolism, pp. 1-33, Plenum Press, New York. 

Nevertheless, genetic engineering of a key enzyme in a chosen pathway does not always result in the enhancement of the end product thus, overexpression of pmt gene carried out in A. belladonna resulted in a 5-fold increased in pmt transcript level but an unchanged tropane alkaloid profile (hyoscyamine and scopolamine), as well as the tropane alkaloids biosynthetic precursors tropine, pseudotropine and tropinone which were not affected [134-135], Similarly, the overexpression of tobacco pmt gene in Dubosia hybrid hairy root cultures produced amounts of tropane alkaloid similar to control roots [131, 136]. All these results seem to indicate the presence of different points of control in the tropane alkaloid metabolic pathway. 

Fig. 2. Alkaloid biosynthetic pathways are associated with a diverse variety of cell types. The tissue-specific localization (shaded) of enzymes and/or gene transcripts are depicted for the biosynthesis of tropane alkaloids in Atropa belladonna and Hyoscyamus niger roots (A), monoterpenoid indole alkaloids in Catharanthus roseus leaves (B), pyrrolizidine alkaloids in Senecio vernalis roots (C), pyrrolizidine alkaloids in Eupatorium cannabinum roots (D), benzyl-isoquinoline alkaloids in Papaver somniferum vascular bundles (E), and protoberberine alkaloids in Thalictrwn flamtm roots (F).

The biosynthetic pathway to both nicotine (5) and the tropane alkaloids includes A-methylputrescine (4) as a probable intermediate. New results6 obtained for nicotine (5) and scopolamine (6) with 1 l-13C,14C wef/zy/awmo-15Nl-A-methyl putrescine 1(4) labels as shown nicely confirm this. The specific incorporation of both stable isotopes was closely similar to that of the 14C label in both alkaloids, indicating intact incorporation of the precursor. The labelling patterns deduced are illustrated ( = 13C, = 15N), and they are in accord with earlier results that were... 

Figure 7.4 Biosynthetic pathway for tropane alkaloids showing the location of enzymes for which the corresponding cDNAs have been isolated.

The usefulness of GC-MS analysis for biosynthetic studies was demonstrated by Patterson and O Hagan [74] in their investigation of the conversion of littorine to hyoscyamine after feeding transformed root cultures of Datura stramonium with deuterium-labeled phenyllactic adds. This study complements previous investigations on the biosynthesis of the tropate ester moiety of hyoscyamine and scopolamine [75], where GC-MS played a key role. It also has general relevance in the biosynthetic pathway of tropane alkaloids in the entire plant kingdom [76]. 

In fhe early 1980s, root cultures of Nicotiana, Hyoscyamus, Datura and Duboisia species were found fo give high yields of nicotine and tropane alkaloids and have proved useful fools for recent studies of the biosynthetic pathways to these alkaloids. Genetically transformed and untransformed root cultures have been generated and used as models for biosynthetic studies (Rhodes et al, 1990 Robins et al, 1994a,b Wildi and Wink, 2002). 

Tropane alkaloid biosynthesis has been studied at the biochemical level, and several enzymes from the biosynthetic pathway have been isolated and cloned, although the pathway has not been elncidated completely at the genetic level (Fig. 3b) (138). L-arginine is converted to the nonproteogenic amino acid L-omithine by the nrease enzyme arginase. Ornithine decarboxylase then decarboxylates ornithine to yield the diamine pntrescine. In Hyoscyamus, Duboisia, and Atropa, putrescine serves as the common precnrsor for the tropane alkaloids. 

Fig. 16. Possible biosynthetic pathway of tropane alkaloids in Hyoscyamus albiis hairy roots transformed with A. rhizogenes MAFF 03-01724.

Fuller details on the incorporation of phenyllactic acid (25) into tropane alkaloids have been published." This acid is a better precursor than phenylalanine for the acid fragments of (20), (21), and (22). The significance of this difference is doubtful it may be the result of several causes, the simplest being more effective diversion of phenylalanine into other biosynthetic pathways. None the less phenyllactic acid (25) is clearly a precursor for tropic acid (19) and atropic acid [as (22)] since it is specifically incorporated and moreover labels the alkaloids in the same way as phenylalanine does. 

Both alkaloids have (+) and (-) forms but only the (-) hyoscyamine and (-) scopolamine are active. The biosynthetic pathway of tropane alkaloids, Fig. (1) is not totally understood, especially at the enzymatic level. Edward Leete has pioneered the biosynthetic studies of tropane alkaloid since 1950"s using whole plants and isotope labels [85-86]. The tropane alkaloid hyoscyamine is bioconverted by the enzyme H6H (hyoscyamine 6p-hydroxylase, EC 1.14.11.11) to scopolamine via 6p-hydroxyhyoscyamine. Hyoscyamine is the ester of tropine and (S)-tropic acid. The (S)-tropic acid moiety derives from the amino acid L-phenylalanine, while the bicyclic tropane ring derives from L-omithine primarily or L-arginine via tropinone. Tropinone is stereospecifically reduced to form either, tropine which is incorporated into hyoscyamine, or on the other hand into pseudotropine which proceeds to calystegines, a group of nortropane derivates that were first found in the Convolvulaceae family [87]. 

It is now accepted that iV-methyl-A1 -pyrrolinium salt is the common precursor of not only tropane alkaloids but also of the N-methylpyrrolidine ring of nicotine, hygrine and cuscohygrine, as shown in Fig. 4. Purification of several enzymes involved in the tropane alkaloid synthesis and the use of radiolabelled precursors have considerably improved our understanding of the biosynthetic pathway. 

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