Ajmaline pathway

Ruppert, M., Woll, J., Giritch, A., Genady, E., Ma, X. Y. and Stockigt, J. 2005. Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system. Planta, 222(5) 888-898. 

FALKENHAGEN, H., STOCKIGT, J., Enzymatic biosynthesis of vomilenine, a key intermediate of the ajmaline pathway, catalyzed by a novel cytochrome P450-dependent enzyme from plant cell cultures of Rauwoljia serpentina. Z. Naturforsch., 1995, 50C, 45-53. 

Ruppert, M., Xueyan, M. and Stockigt, J. (2005) Alkaloid biosynthesis in Rauvolfia-cDNA cloning of the major enzymes of the ajmaline pathway. Curr. Org. Chem., 9, 1431-44. 

In the next step of the ajmaline pathway, vinorine synthase transforms the sarpagan alkaloid epi-vellosamine to the ajmalan alkaloid vinorine (84). Vinorine synthase also has been purified from Rauwolfla cell culture, subjected to protein sequencing, and cloned from a cDNA library (85, 86). The enzyme, which seems to be an acetyl transferase homolog, has been expressed heterologously in E. coli. Crystallization and site-directed mutagenesis studies of this protein have led to a proposed mechanism (87). 

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system. Planta 2005 222 888-898. 109. 

A number of side routes originate from various intermediates of the ajmaline pathway (AP) are shown in Figure 22. They lead to a complex biosynthetic network of sarpagan and ajmalan alkaloids in Rauvolfia. 

Within the natural products field, the investigation of complete biosynthetic pathways at the enzyme level has been especially successful for plant alkaloids of the monoterpenoid indole alkaloid family generated from the monoterpene gluco-side secologanin and decarboxylation product of tryptophan, tryptamine [3-5]. The most comprehensive enzymatic data are now available for the alkaloids ajmalicine (raubasine) from Catharanthus roseus, and for ajmaline from Indian Rauvolfia serpentina [6] the latter alkaloid with a six-membered ring system bearing nine chiral carbon atoms. Entymatic data exsist also for vindoline, the vincaleucoblastin (VLB) precursor for which some studies on enzymatic coupling of vindoline and catharanthine exist in order to get the so-called dimeric Catharanthus indole-alkaloids VLB or vincristine [7-9] with pronounced anti-cancer activity [10, 11]. 

The biosynthetic pathways of reserpine and other alkaloids of Rauwalfia have been extensively studied by several authors. Leete (l8, 19) fed DL-[2- C]-tryptophan into Rauwolfia serpentina (3 years old plants) which led to the formation of radioactive ajmaline, serpentine and reserpine (18). Later, he found that radioactive serpentine was labeled solely at C-5 indicating that tryptophan was a direct precursor of the -carboline moiety of this alkaloid (19). Other radioactive precursors have been administered to Rauwolfia plants such as [l- C] acetate (20), [2- C] acetate (21), [2-11 C] alanine (20) and [2-l2 C] glycine (21, 22). All incorporated into ajmaline and reserpine. 

R. serpentina cell cultures, raucaffricine levels amounted to 1.2 g/L medium, whereas ajmaline levels reached only 0.3 g/L medium (Schtibel et al, 1986). It is interesting to note that raucaffricine is a fypical constituent of R. cajfra, but has not been isolated from other Rauvolfia species. However, in Rauvolfia cell cultures the compound is found in all species tested, with a maximum yield in R. serpentina. In other words, under these growth conditions, the pathway to ajmaline appears to have become deregulated. 

Stockigt, J., Panjikar, S., Ruppert, M., Barleben, L., Ma, X., Loris, E. and Hill, M. (2007) The molecular architecture of the major enzymes from ajmaline biosynthetic pathway. Phytochem. Rev., 6,15-34. 

The biosynthetic pathway for ajmaline in R. serpentina is one of the best-characterized terpenoid indole alkaloid pathways. Much of this progress has been detailed in a recent extensive review (78). Like all other terpenoid indole alkaloids, ajmaline, an antiarrhythmic drug with potent sodium channel-blocking properties (79), is derived from deglycosylated strictosidine (Fig. 2c). 

After deglucosylation, the pathway proceeds through a 4,21-Dehydrogeissoschizine derivative to ajmalicine (an a-Blocking spasmolytic agent, used for tinnitus and cranial trauma with an ergot derivative). If cyclization occurs between C-17 and C-18, the yohimbine nucleus is produced, whose derivatives include the Rauvolfia alkaloids reserpine and resdnnamine (antihypertensive activity). Ajmaline, formerly used as an antiarrythmic, also occurs in Rauvolfia species, and several of the enzymes in the pathway have been isolated. Recent considerations suggest that the C-16-C-5 bond may be formed before the N-4-C-21 bond (Fig. 38). 

Biosynthesis R. is derived from vomilenine, an intermediate in the biosynthetic pathway to the antiarrhyth-mic ajmaline, by glucosylation. The corresponding glucosyl transferase is membrane-bound, dependent on uridine diphosphate glucose, and has a high substrate specificity. A soluble, substrate-specific glu-cosidase effects the reversal of this reaction see also Rauvolfia alkaloids. 

The complexity of the ajmaline structure makes it difficult to identifying its biosynthetic pathway. The solution to this problem is an in-depth elucidation of each reaction step and each enzyme involved in the catalysis." This article reviews the relevant research on the ajmaline s main biosynthetic pathway and its side routes, the route beyond ajmahne, the application of in vivo NMR, the chemo-enzymatic significance of the involved enzymes and, their reaction mechanisms and the enzyme X-ray crystal stmctures. 

Figure 6 The main biosynthetic pathway of ajmaline is shown ( indicates the cDNAs of enzymes which have been functionally, heterologously expressed).

Final stage of the pathway to ajmaline is also conducted hy an esterase, named acetyl-ajmalan esterase, or acetylajmahne esterase (AAE) or 2p(R)-170-acetylajmalan acetylesterase. The systematic name for AAE is 170-acetylajmahne O-acetyl-hydrolase. ... 

By this control function, acetylajmalan esterase obviously directs the pathway to the final ajmalan-type structure, the alkaloid ajmaline (Figure 20). 

Figure 22 Side routes of the biosynthetic pathway of ajmaline ( indicates the enzymes (cDNAs) which have been heterologously expressed).

A side route may also take place at the end of the ajmahne pathway by meta-boUsm of ajmaline (1) itself By administering ajmaline (1) to the Rauuolfia suspended cells in high concentrations (up to 1 g/L), it is converted to a small novel group of six derivatives named raumaclines (Figures 34 and 39). These new alkaloids were, however, detected in the surrounding nutri-tion medium. This bioconversion can be summarized as Figure 34. 

The enzyme-catalyzed biosynthesis of the sarpagan-ajmalan-type firni-ihes of monoterpenoid indole alkaloids in the genus Rauvolfia, in particular R. serpentina Benth. ex Kurz, have recendy been delineated in detail. The major pathway leading to the structural intricate antiarrhythmic ajmaline, together with several side routes and bypasses, form a comprehensive and complex anabolic network of which a multitude of single-enzymatic... 

Plant Cells. The metabolism of unlabelled alkaloids in plant cell cultures has been followed using heteronuclear multiple quantum coherence NMR. The addition of ajmaline to cell suspension cultures of somatic hybrids of Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne (R x R cells) resulted in its conversion to raumacline. When, vinorine, an intermediate of ajmaline metabolism, was added to suspensions on R x R cells, vellosimine was formed and then converted to 10-deoxysarpagine. However, vinorine was apparently metabolised by a second pathway to vomilenine or raucaffricine." ... 

---