[Show abstract][Hide abstract] ABSTRACT: The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta. The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine. An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration. Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity. SDS-PAGE analysis showed a Mr for the enzyme of approximately 50 kDa. The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced. The enzyme preparation (> 875-fold enrichment) delivering the N-terminal sequence was isolated from R. serpentina cell suspensions. Sequence alignment of the five peptides showed highest homologies in a range of 30-71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis. Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.
[Show abstract][Hide abstract] ABSTRACT: Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry. This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R. serpentina roots, R. serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco. The influence of buffer composition and analyte structures on separation is discussed.
Journal of Chromatography A 09/2002; 967(1):85-113. · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Natural Products 08/2002; 65(7):1006-10. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new monoterpenoid indole alkaloid, 10-hydroxy- N(alpha)-demethyl-19,20-dehydroraumacline ( 1), was isolated as a mixture of E- and Z-isomers from hairy root culture of Rauvolfia serpentina Benth. ex Kurz (Apocynaceae) and the structure was determined by 1D and 2D NMR analyses. The new indole alkaloid represents the first naturally occurring alkaloid of the raumacline group and its putative biosynthetical pathway is discussed.
[Show abstract][Hide abstract] ABSTRACT: 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 (Nbeta-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.
European Journal of Biochemistry 05/2002; 269(8):2204-13. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new monoterpenoid indole alkaloid, 3-oxo-rhazinilam (1), was isolated from intergeneric somatic hybrid cell cultures of Rauvolfia serpentina and Rhazya stricta, and the structure was determined by detailed 1D and 2D NMR analysis. It was also proved that 3-oxo-rhazinilam (1) is a natural constituent of the hybrid cells.
Journal of Natural Products 02/2001; 64(1):114-6. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An efficient system for the analysis of indole alkaloids by HPLC on a reversed-phase column using an ion pair technique is described. The optimised chromatographic conditions allowed the successful separation of 22 standard monoterpenoid indole alkaloids (including some isomers) and tryptamine. The described HPLC system was applied to the analysis of alkaloids in intergeneric somatic hybrid cell cultures of Rauvolfia serpentina x Rhazya stricta.
[Show abstract][Hide abstract] ABSTRACT: The alkaloid pattern in Rauvolfia serpentina x Rhazya stricta somatic hybrid cell subcultures R x R17 K was studied and 11 compounds were identified on the basis of their spectral data. Among them, 1,2-dehydroaspidospermidine, rhazinilam, stemmadenine and tabersonine were reported as typical of Rhazya species while vomilenine and sarpagine are characteristic of Rauvolfia alkaloid metabolism. The alkaloid pattern in R x R17 K subcultures was compared with that in the other hybrid cell subcultures (R x R17 M) which were developed from the same origin hybrid cultures but have been maintained separately for about ten years. The data presented here exhibit pronounced divergence of the alkaloid patterns in R x R17 K and R x R17 M cell subcultures.
[Show abstract][Hide abstract] ABSTRACT: Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase, known as raucaffricine-O-β-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/μg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/μg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.
[Show abstract][Hide abstract] ABSTRACT: The treatment of Rauwolfia serpentina x Rhazya stricta somatic hybrid cell suspension culture with 100 μM of methyl jasmonate led to a general increase in indole alkaloid content and to qualitative changes in the alkaloid pattern.
The content of Six alkaloids were investigated with respect to their content in both the cell biomass and nutrition medium.
Intracellular 17-O-acetyl-norajmaline content on the 5th day after treatment had increased about 40-fold compared with the control culture.
The respective concentrations of the other alkaloids increased by a factor of two to five. In total 26 indole alkaloids were
identified in extracts of the methyl jasmonate-treated culture by TLC, UV, MS and NMR data and comparison with reference alkaloids.
The identification of macrophylline, yohimbine oxindole and yohimbine pseudoindoxyl has not been reported before in Rauwolfia serpentina or Rhazya stricta plants nor in cell cultures derived from these plants.