Search results for "Rauvolfia serpentina"
showing 10 items of 29 documents
Expression, purification, crystallization and preliminary X-ray analysis of strictosidine glucosidase, an enzyme initiating biosynthetic pathways to …
2005
Abstract Strictosidine β- d -glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 and PEG 4000 (10%) as precipitant buffer. The crystals of strictosidine glucosidase belong to the space group P 42 1 2 with unit cell dimensions of a =157.63, c =103.59 A and diffract X-rays to 2.48-A resolution.
Deoxysarpagine Hydroxylase — A Novel Enzyme Closing a Short Side Pathway of Alkaloid Biosynthesis in Rauvolfia
2002
Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase…
Glucosylation of isatin-3-oxime followed by 2D in situ NMR in plant cells at highest magnetic field without labelling.
2001
The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gradient assisted NMR spectroscopy in plant cell suspension cultures of Rauvolfia serpentina without labelling. The applied high magnetic field of 800 MHz allowed measurements within 20 min at concentrations of 1 of 5.76 mM. Complete glucosylation of 1 occurs inside the cells within 72 hours. During this time isatin-3-oxime-glucoside (2) accumulates without further metabolism.
Heterologous expression of aRauvolfiacDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids
2002
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…
High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic–electrospray ionisation mass spectrometric analysis …
2002
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 w…
Development of an efficient system for the separation of indole alkaloids by high performance liquid chromatography and its applications
2001
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.
Induction of alkaloid diversity in hybrid plant cell cultures
1999
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 a…
3-Oxo-rhazinilam: A New Indole Alkaloid from Rauvolfia serpentina × Rhazya stricta Hybrid Plant Cell Cultures
2000
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.
Isolation and Structure Elucidation of a New Indole Alkaloid from Rauvolfia serpentina Hairy Root Culture: The First Naturally Occurring Alkaloid of …
2002
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.
In vivo monitoring of alkaloid metabolism in hybrid plant cell cultures by 2D cryo-NMR without labelling
2003
Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne were carried out. When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13C. Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800). Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe that the alkaloid enters two metabolic rou…