Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus

The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline.     

Phytochrome is involved in the light-regulation of vindoline biosynthesis in catharanthus

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyl-transferase (DAT) catalyzes the final step in the biosynthesis of the monoterpenoid indole alkaloid, vindoline. Previous studies have shown that the appearance of DAT activity in etiolated seedlings of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme activity is restricted principally to the cotyledons. Evidence is now presented that phytochrome is involved in the light-mediated induction of DAT activity in Catharanthus cotyledons.     

Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots

Madagascar periwinkle [Catharanthus roseus (L.) G Don] is a pantropical plant of horticultural value that produces the powerful anticancer drugs vinblastine and vincristine that are derived from the dimerization of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine. The present study describes the genetic engineering and expression of the terminal step of vindoline biosynthesis, deacetylvindoline-4-O-acetyltransferase (DAT) in Catharanthus roseus hairy root cultures. Biochemical analyses showed that several hairy root lines expressed high levels of DAT enzyme activity compared to control hairy root cultures expressing β-gulucuronidase activity (GUS) activity. Metabolite analysis using high performance liquid chromotagraphy established that hairy root extracts had an altered alkaloid profile with respect to hörhammericine accumulation in DAT expressing lines in comparison to control lines. Further analyses of one hairy root culture expressing high DAT activity suggested that DAT expression and accumulation of hörhammericine (9) were related. It is concluded that expression of DAT in hairy roots altered their MIA profile and suggests that further expression of vindoline pathway genes could lead to significant changes in alkaloid profiles. Evidence is provided that hörhammericine (9) accumulates via a DAT interaction with the root specific minovincinine-19-O-acetyltransferase (MAT) that inhibits the MAT mediated conversion of hörhammericine (9) into 19-O-acetyl-hörhammericine (12).     

Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures

A callus strain with stable ability for vindoline synthesis was selected from many prepared Catharanthus roseus leaf calli to study the regulation of vindoline biosynthesis as well as other indole alkaloids. It was shown that light and plant growth regulators significantly influenced the biosynthesis of vindoline and other alkaloids as well as acidic and basic peroxidase activities. Light promoted vindoline and serpentine biosynthesis, and stimulated plastid development and peroxidase activity. However, 2,4-D suppressed the biosynthesis of all indole alkaloids and peroxidase activity. Our results suggest that light or plant hormones regulate vindoline, serpentine and other alkaloid biosynthesis and accumulation by influencing peroxidase activity and the differentiation status of callus cultures, especially chloroplast development. Some possible relationships between serpentine or vindoline biosynthesis and peroxidase activity are proposed.     

Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus plants

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.     

Vindoline formation in shoot cultures of Catharanthus roseus is synchronously activated with morphogenesis through the last biosynthetic step

Background and Aims

The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here.

Methods

Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle.

Key Results

In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins.

Conclusions

The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.Key words: Catharanthus roseus, deacetylvindoline acetyl CoA acetyltransferase, DAT, in vitro shoot cultures, morphogenesis, vindoline     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus

The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.     

A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus

Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation. These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light. The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway. However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C. roseus cell-suspension cultures. These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms.     

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P-450 dependent monooxygenase.

An end-product indole alkaloid, catharanthine, inhibits a membrane-bound cytochrome P-450 dependent monooxygenase of the higher plant, $\text{Vinca rosea}$. Kinetic analysis revealed the alkaloid to be a reversible, linear, non-competitive inhibitor (K_i=1 mM) with respect to its substrates, geraniol and NADPH. Comparable inhibition of the solubilized monooxygenase by catharanthine tends to exclude a mechanism based upon disruption of membrane organization. On the basis of its inhibition of solubilized hydroxylase in the presence and absence of sodium cholate, it is also unlikely that catharanthine competes for putative phospholipid binding site(s). Two additional end-product alkaloids, vinblastine and vindoline were less inhibitory. Since the hydroxylase catalyzes one of the first committed steps in the biosynthesis of indole alkaloids, these observations suggest feedback control of the pathway by catharanthine.     

Auxins induce tryptophan decarboxylase activity in radicles of catharanthus seedlings

Germinating seedlings of Catharanthus roseus produce monoterpenoid indole alkaloids as a result of a transient increase of tryptophan decarboxylase (TDC) activity. The influence of auxins on this transient rise of TDC activity was studied. External application of indolebutyric acid or 2,4-dichlorophenoxyacetic acid at a concentration of 20 to 40 μm enhanced and prolonged the rise in TDC activity in developing seedlings. Auxin treatment also influenced the morphology of the seedlings; it induced a shortening and thickening of the hypocotyl and the radicle and promoted the initiation of lateral roots in the radicle. During development, the radicles of auxin-treated seedlings displayed a gradual increase in TDC activity that was absent in the radicles of untreated controls. Examination of immunoblots revealed anti-TDC reactive proteins in extracts from radicles of auxin-treated seedlings, but none in extracts from radicles of control seedlings. In contrast, TDC activity and immunoreactive protein levels in the aerial parts of controls and auxin-treated seedlings were comparable. Our results indicate that externally applied auxins induce both abnormal development and TDC activity in the radicles of Catharanthus seedlings. Although auxins slightly delayed the light-mediated induction of the cotyledon-specific last step in vindoline biosynthesis (i.e. acetylcoenzyme A: deacetylvindolin-O-acetyltransferase activity), seedlings still synthesized vindoline, one of the major alkaloid end products.     

The effects of phenobarbital and ketoconazole on the alkaloid biosynthesis in Catharanthus roseus cell suspension cultures

The cytochrome P450 enzyme geraniol 10-hydroxylase plays an important role in the biosynthesis of terpenoid indole alkaloids in suspension cultures of Catharanthus roseus. The activity of this enzyme was induced by the treatment of cells with phenobarbital, and inhibited by treatment with ketoconazole. The alkaloid accumulation increased after phenobarbital treatment whereas it decreased after ketoconazole treatment. Phenobarbital and ketoconazole did not affect the in vivo conversion rate of loganin to secologanin, a reaction proposed to be catalyzed by a cytochrome P450 enzyme.     

The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

The biological activity of monoterpenoid indole alkaloids (MIAs) has led to their use in cancer treatment and other medical applications. Their biosynthesis has involved the formation of reactive intermediates by responsible enzymes to elaborate several different chemical scaffolds. Modification of scaffolds through different substitution reactions has produced chemically diverse MIAs and related biological activities. The present study characterizes the three‐step pathway involved in the formation of (+)‐echitovenine, the major O‐acetylated MIA of Catharanthus roseus roots, and differentiates it from a parallel pathway involved in the formation of hörhammericine. Separate hydrolases convert a common reactive MIA intermediate to aspidosperma skeletons of opposite specific rotations, that is (+)‐vincadifformine and (?)‐tabersonine, respectively. The formation of (+) minovincinine from (+) vincadifformine 19‐hydroxylase (V19H) is catalyzed by a root‐specific cytochrome P450 with high amino acid sequence similarity to the leaf‐specific tabersonine‐3‐hydroxylase involved in vindoline biosynthesis. Similarly, O‐acetylation of (+)‐minovincinine to form (+) echitovenine involves minovincinine‐O‐acetytransferase. The substrate specificity of V19H and MAT for their respective (+)‐enantiomers defines the separate enantiomer‐specific pathway involved in (+)‐echitovenine biosynthesis and differentiates it from a parallel (?)‐enantiomer‐specific pathway involved in the formation of hörhammericine from (?)‐tabersonine.