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R Verpoorte

Publications and source records attributed to R Verpoorte.

At least 55 records · Page 3Linked to original sources
PubMed1998

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Alkaloids↗
PubMed1998

4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates.

Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.

Acyltransferases↗
PubMed1998

Cardiovascular activity of the crude alkaloidal fraction from Tabernaemontana pandacaqui in the rat.

The effects of a crude alkaloidal (CA) fraction from the stem of Tabernaemontana pandacaqui Poir. on the blood pressure and heart rate were investigated in conscious as well as anesthetized rats. The CA fraction exerted a hypotensive activity in both experimental models. In pentobarbital anesthetized rats, an intravenous administration of the CA fraction caused two consecutive hypotensive and bradycardiac responses. In order to investigate the mechanism of the responses, the effect of the CA fraction on the blood pressure and the heart rate was tested in various experimental animals such as pithed rats, reserpinized rats under pentobarbital anesthesia and atropine- or chlorpheniramine-treated rats under pentobarbital anesthesia. The results obtained suggest that the hypotensive and bradycardiac responses of the first phase might involve cholinergic and central mechanisms, whereas those of the second phase involve mechanisms which are mediated by central, biogenic amines, acetylcholine and histamine.

Adjuvants, Anesthesia↗
PubMed1998

Neuropharmacological activities of the crude alkaloidal fraction from stems of Tabernaemontana pandacaqui Poir.

The crude alkaloidal (CA) fraction from the stem of Tabernaemontana pandacaqui Poir. was studied for its pharmacological activity on the central nervous system (CNS) of animals. The CA fraction was found to produce symptoms of CNS depression in conscious rats and mice, viz. reduction in spontaneous motility, potentiation of pentobarbital sleeping time, prolongation of latency of convulsions induced by pentylenetetrazole and antinociception. However, the fraction could not antagonize oxotremorine-induced tremor. The observations suggest that the CA fraction possesses a CNS depressant activity.

Alkaloids↗
PubMed1998

Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation.

We have created a population of transgenic tobacco plants carrying cDNAs encoding two consecutive enzymes from early stages in monoterpenoid alkaloid biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan decarboxylase (tdc) and strictosidine synthase (str1) together with a selectable marker gene, were introduced on a single transforming plasmid into tobacco leaves by particle bombardment. Analysis of 150 independent transgenic plants at the DNA and RNA levels demonstrated a range of integration events and steady-state transcript levels for the tdc and str1 transgenes. Southern blot analysis indicated that the tdc and str1 transgenes were integrated at least once in all 150 transformants giving a 100% co-integration frequency of the two unselected genes carried on the same plasmid. A comparison of Southern and northern data suggested that in 26% of the plants, both tdc and str1 transgenes were silenced, 41% demonstrated a preferential silencing of either the tdc or the str1 transgene, with the remaining 33% of the plants expressing both transgenes. We observed no clear correlation between the number of integration events of a specific transgene and the levels of accumulated transcript. Twenty plants representing the range of molecular diversity in the transgenic population were selected for further analysis. Seeds were collected from self-fertilised transformants and germinated on medium containing kanamycin. Seedlings were harvested after 7 weeks and TDC and STR1 enzymatic assays were carried out. We observed a 24- and 110-fold variation in levels of TDC and STR1 activities, respectively. Our data correlate molecular diversity with biochemistry and accumulation of end-product and provide a detailed molecular and biochemical characterization of transgenic plants transformed with a single plasmid carrying two genes of secondary metabolism.

Alkaloids↗
PubMed1997

Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.

In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor-treated C. robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM. Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform. The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.

Anthraquinones↗
PubMed1995

Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms.

Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. All strictosidine synthase isoforms were found to be glycoproteins. The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures. The ratio of the different isoforms differed slightly between these sources. The kinetic parameters of the isoforms showed no significant differences. The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far. It was demonstrated that the apparent Michaelis constant for tryptamine (approx. 9 microM) is much lower than values reported previously. The presence of weak product inhibition (Kp approx. 35 times Km) was established, whereas substrate inhibition was not detected.

Carbon-Nitrogen Lyases↗
PubMed1995

Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

Alkaloids↗
PubMed1993

Purification and characterization of anthranilate synthase from Catharanthus roseus.

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 +/- 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 +/- 5 kDa. The enzyme had a pI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH4+ as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg2+, Km values for L-glutamine was determined to be 0.37 +/- 0.05 mM, for chorismate 67 +/- 3 microM, and for MgCl2 0.26 +/- 0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperatively of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20 microM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperatively for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.

Anthranilate Synthase↗
PubMed1993

Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy.

Cytochrome P-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. An efficient PCR strategy for isolating cytochrome P-450 cDNA clones from plant cDNA libraries is described. A set of degenerate primers for PCR amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome P-450 proteins. Using this primer set and a non-specific primer, complementary to either the poly(A) tail of the cDNA clones or a phage vector sequence, we isolated 16 different cytochrome P-450 cDNA sequences from a cDNA library of Catharanthus roseus.

Amino Acid Sequence↗
PubMed1993

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density.

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in mumoles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high- and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.

Acyclic Monoterpenes↗
PubMed1993

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants.

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 monooxygenases, was purified from a cell suspension culture of the higher plant Catharanthus roseus. Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identify of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animals species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.

Amino Acid Sequence↗
PubMed1992

Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors.

Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. The complete mRNA sequence for the enzyme strictosidine synthase (SSS) was determined. Comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified SSS indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. SSS is encoded by a single-copy gene indicating that isoenzymes reported by others are formed post-translationally from a single precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding another enzyme essential for indole alkaloid biosynthesis, are coordinately regulated. In plants steady-state mRNA levels are highest in roots. In cell suspension cultures the genes are rapidly down-regulated by auxin. In contrast, both genes are strongly induced by fungal elicitors such as Pythium aphanidermatum culture filtrate or yeast extract. Induction is a rapid, transcriptional event occurring independent of de novo protein synthesis. These results show that a first important regulatory step in the complex process leading to indole alkaloid accumulation in C. roseus suspension cells is transcription of the biosynthetic genes.

Alkaloids↗
PubMed1992

The influence of tryptophan and tryptamine feeding and light on alkaloid biosynthesis in cinchona seedlings.

It has previously been shown that, at the onset of germination of CINCHONA seeds, transient increases in both the tryptophan level and tryptophan decarboxylase (TDC) activity occur. Subsequently, in the seedlings the levels of the TDC product tryptamine and its derived alkaloids increase. We investigated whether this process can be influenced by external tryptophan or tryptamine feeding. Besides, the possible role of light in alkaloid biosynthesis was studied by monitoring this process in etiolated seedlings. Tryptophan feeding slightly raised the TDC activity in the seedlings, but alkaloid production remained unaltered. During tryptamine feeding, the transient increases in tryptophan level and TDC activity were still observed, and also here, alkaloid production remained unaltered. Finally, in etiolated seedlings the alkaloid biosynthetic pathway proceeded normally. Thus, in germinating CINCHONA seedlings alkaloid production is not susceptible to tryptophan or tryptamine feeding and independent of light.

Journal Article↗
PubMed1992

Search for Factors Related to the Indole Alkaloid Production in Cell Suspension Cultures of Tabernaemontana divaricata.

Three strains derived from one cell line of a suspension culture of TABERNAEMONTANA DIVARICATA were obtained by subculturing on three different media: (i) Strain A: normal MS-medium (1), (ii) Strain S: medium in which the carbon source was starch instead of sucrose, and (iii) Strain N: medium in which the ammonium/nitrate ratio was changed from 1: 2 to 1:1. The alkaloid contents of all three strains were compared at each subculture for nearly one year. Strain N showed after its initiation a gradually increasing alkaloid production up to levels of about 500 microg/gDW. Strain A (on the original medium) showed a stable, but low alkaloid production (+/- 20 microg/gDW) while strain S turned into a non-producing line. The dissimilation curves, morphology, intracellular carbohydrates, and free amino acid pools of all three strains were determined. Strain N showed the highest biomass, the least dissimilation, most plastids, most intracellular carbohydrates, and high levels of arginine and glutamine, while strain S showed the lowest biomass, most dissimilation, no plastids, little intracellular carbohydrates, and high levels of arginine, phenylalanine, and tyrosine. The observed differences are discussed and evidence is provided that the differences are not caused by genetic instability.

Journal Article↗
PubMed1991

Thermospray Liquid Chromatography/Mass Spectrometry (TSP LC/MS) Analysis of the Alkaloids from Cinchona in vitro Cultures.

The alkaloids from CINCHONA LEDGERIANA shoot cultures and from CINCHONA ROBUSTA shoot cultures and a compact globular structure (CGS) culture were analyzed by thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Because of the relative stability of the alkaloids under TSP discharge ionization conditions, a protonated molecule was observed in the mass spectra with hardly any fragmentation. When the reference compounds were available, the knowledge of the molecular mass and of the retention time was sufficient to identify most of the alkaloids. HPLC with UV photodiode-array detection complemented LC/MS perfectly by providing information about the aromatic part of the alkaloids (structure and substitution pattern). New alkaloids detected in CINCHONA IN VITRO cultures were 5-methoxytryptamine and corynantheal. In order to determine whether 5-methoxytryptamine was a precursor of the methoxylated quinolines, this indole was incubated with secologanin and several CINCHONA ROBUSTA crude protein extracts. Under all conditions tested, the coupling of 5-methoxytryptamine with secologanin remained unsuccessful. Only tryptamine condensed with secologanin to yield strictosidine. These results indicate that CINCHONA cells are able to methoxylate simple indoles like tryptamine and that 5-methoxytryptamine is very likely not used for the subsequent biosynthesis of the methoxylated quinolines.

Journal Article↗