Three new monoterpenoid indole alkaloids, 19(S),20(R)-dihydroperaksine (1), 19(S),20(R)-dihydroperaksine-17-al (2), and 10-hydroxy-19(S),20(R)-dihydroperaksine (3), along with 16 known alkaloids 4-19 were isolated from hairy root culture of Rauvolfia serpentina, and their structures were elucidated by 1D and 2D NMR analyses. Taking into account the stereochemistry of the new alkaloids and results of preliminary enzymatical studies, the putative biosynthetical relationships between the novel alkaloids are discussed.

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...

No abstract

No abstract