Indirect Identification of 4,21–Dehydrocorynantheine Aldehyde as an Intermediate in the Biosynthesis of Ajmalicine and Related Alkaloids

Stöckigt, Joachim; Rueffer, Martina; Zenk, Meinhart H.; Ga, Hoyer

doi:10.1055/s-0028-1097376

Cited by 31 publications

(11 citation statements)

References 6 publications

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“…This result supports the identification of (8) as the end product of the cell-free strictosidine deglucosylation, as the reduction of (8) leads to tetrahydroalstonine. When the concentration of NaBH 3 CN was increased to a 2000-fold excess, the two products sitsirikine (10) and isositsirikine (11) were identified, which demonstrates that 4,21-dehydrocorynantheine aldehyde (7) is involved in the indole alkaloid biosynthesis in R. serpentina as well as it has been shown earlier for C. roseus [42].…”

Section: Products Of Enzymatic Deglucosylation Of Strictosidinesupporting

confidence: 71%

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Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Gerasimenko

Sheludko

et al. 2002

European Journal of Biochemistry

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Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nb-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.Keywords: strictosidine b-D-glucosidase; heterologous expression; Rauvolfia serpentina; ajmaline; indole alkaloid biosynthesis.Elucidation of the biosynthesis of natural plant products has been a matter of investigation for over half a century. Although there have been major efforts in the field, only very few biosynthetic pathways have been delineated in detail at the enzymatic level. Knowing the enzymes involved is, however, a prerequisite for understanding the biosynthetic mechanisms. The next steps are to search for the participating genes and to clarify the regulation of product synthesis, with the aim of influencing the biosynthesis on a rational basis. The best known pathways comprise those of the flavonoid biosynthesis [1,2], the isoquinoline alkaloid formation [3,4] and the biosynthesis of indole alkaloids [5,6].The key intermediate in the biosynthesis of all monoterpenoid indole alkaloids is the glucoalkaloid strictosidine [7][8][9][10]. It is formed by condensation of tryptamine, the decarboxylation product of tryptophan, and the monoterpene secologanin catalysed by the enzyme strictosidine synthase (SS) [11]. The biosynthetic pathways leading to different classes of indole alkaloids branch off somewhere downstream of strictosidine. The first point where this divergence may take place is the deglucosylation of strictos...

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Section: Products Of Enzymatic Deglucosylation Of Strictosidinesupporting

confidence: 71%

“…5). Similar experiments have been previously carried out with rather crude enzyme extracts from C. roseus cell suspensions [42]. To gain more detailed insight into the mechanism of strictosidine conversion, we carried out a series of experiments.…”

Section: Products Of Enzymatic Deglucosylation Of Strictosidinementioning

confidence: 96%

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Gerasimenko

Sheludko

et al. 2002

European Journal of Biochemistry

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“…Incubation with [methyl-'4C/methionine (13). Crude enzyme solution (24 ml) was incubated with FAD (0.86 mg), NADPH (1.05 mg).…”

Section: Experimental Partmentioning

confidence: 99%

Biosynthesis of the Indole Alkaloids. Cell‐free Systems from Catharanthus roseus Plants

Kutney

Choi

Honda

et al. 1982

Helvetica Chimica Acta

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SummaryCell-free systems from Catharunthus roseus plants are utilized for various studies relating to the biosynthesis of indole alkaloids. Tryptamine (5) and secologanin (6), two fundamental building units, are shown to be incorporated into the alkaloid vindoline (7). In another study, catharanthine (18) and vindoline (7) are utilized by this enzyme system and coupled to the important bisindole biointermediate 3', 4'-anhydr~vinblastine~) (17). The latter substance is, in turn, incorporated and converted to the natural alkaloids leurosine (8), Catharine (9) and vinblastine (lo), thereby providing information about the biosynthesis of these complex molecules. High pressure liquid chromatography assay of the enzymic mixture sheds light on the enzymes involved in the coupling of 18 and 7.The utilization of cell-free systems from Catharanthus roseus seedlings, plants or tissue cultures in indole alkaloid biosynthesis has been the subject of a number of recent publications from several laboratories. Scott & Lee [S] were the first to describe a cell-free system capable of synthesizing ajmalicine (1) and geissoschizine (4) from tryptamine (5) and secologanin (6; s. below). Subsequent studies in their or other laboratories [6-181 have provided information on some of the stages relating to the biosynthesis of such CorynanthP type alkaloids. For example, the condensation of tryptamine and secologanin for the biosynthesis of 1, 4 and the related alkaloids 19-epiajmalicine (2) and tetrahydroalstonine (3) occurs in the presence of NADPH as a co-factor. More recent studies, particularly by Scott, Zenk, Stockigt and their co-workers, have provided detailed information on the later-stage biointermediates [ 181. While these investigations were underway we addressed ourselves to the biosyntheses of Aspidosperma and fboga alkaloids. We wish to describe our experiments concerning the use of cell-free systems to demonstrate certain aspects of the biosynthesis of the alkaloid vindoline (7; s.

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“…The route from the simple precursors tryptamine (or tryptophan) and secologanin via strictosidine to the heteroyohimbine alkaloids was established by STOCKIGT et al [37] (Fig. 2).…”

Section: Discussionmentioning

confidence: 98%

Alkaloid Distribution in Some African Rauvolfia Species

We¹

1983

Planta Med

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Indirect Identification of 4,21–Dehydrocorynantheine Aldehyde as an Intermediate in the Biosynthesis of Ajmalicine and Related Alkaloids

Cited by 31 publications

References 6 publications

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Biosynthesis of the Indole Alkaloids. Cell‐free Systems from Catharanthus roseus Plants

Alkaloid Distribution in Some African Rauvolfia Species

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