Publications

• A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

  Besseau S., Kellner F., Lanoue A., Thamm A.M., Salim V., Schneider B., Geu-Flores F., Hoefer R., Guirimand G., Guihur A., Oudin A., Glevarec G., Foureau E., Papon N., Clastre M., Giglioli-Guivarc'h N., St-Pierre B., Werck-Reichhart D., Burlat V., De Luca V., O'Connor S.E., Courdavault V.

  Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from ... >> More

  Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis. << Less

  Plant Physiol. 163:1792-1803(2013) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Developmental and light regulation of desacetoxyvindoline 4-hydroxylase in catharanthus roseus (L.) G. Don. . Evidence Of a multilevel regulatory mechanism.

  Vazquez-Flota F.A., De Luca V

  The expression of desacetoxyvindoline 4-hydroxylase (D4H), which catalyzes the second to the last reaction in vindoline biosynthesis in Catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. Developmental studies with etiolated and light-treated seedlings ... >> More

  The expression of desacetoxyvindoline 4-hydroxylase (D4H), which catalyzes the second to the last reaction in vindoline biosynthesis in Catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. Developmental studies with etiolated and light-treated seedlings suggested that although light had variable effects on the levels of d4h transcripts, those of D4H protein and enzyme activity could be increased, depending on seedling development, up to 9- and 8-fold, respectively, compared with etiolated seedlings. However, light treatment of etiolated seedlings could stop and reverse the decline of d4h transcripts at later stages of seedling development. Repeated exposure of seedlings to light was also required to maintain the full spectrum of enzyme activity observed during seedling development. Further studies showed that a photoreversible phytochrome appeared to be involved in the activation of D4H, since red-light treatment of etiolated seedlings increased the detectable levels of d4h transcripts, D4H protein, and D4H enzyme activity, whereas far-red-light treatment completely reversed this process. Additional studies also confirmed that different major isoforms of D4H protein exist in etiolated (isoelectric point, 4.7) and light-grown (isoelectric point, 4.6) seedlings, suggesting that a component of the light-mediated activation of D4H may involve an undetermined posttranslational modification. The biological reasons for this complex control of vindoline biosynthesis may be related to the need to produce structures that could sequester away from cellular activities the cytotoxic vinblastine and vincristine dimers that are derived partially from vindoline. << Less

  Plant Physiol 117:1351-1361(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus.

  De Carolis E., De Luca V.

  A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been purified to near homogeneity from Catharanthus roseus. The purification procedure combined conventional chromatographic methods and cosubstrat ... >> More

  A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been purified to near homogeneity from Catharanthus roseus. The purification procedure combined conventional chromatographic methods and cosubstrate affinity chromatography on alpha-ketoglutarate-Sepharose. The specific activity of the 4-hydroxylase was enriched over 2000-fold compared to the crude homogenate with a recovery of 1.6%. The molecular mass of the native and denatured 4-hydroxylase was found to be 45 and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pI values 4.6, 4.7, and 4.8. The enzyme did not require most divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where 2-oxoglutarate is the first substrate to bind followed by the binding of O2 and desacetoxyvindoline. The first product to be released was deacetylvindoline followed by CO2 and succinate, respectively. << Less

  J. Biol. Chem. 268:5504-5511(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isolation and characterization of a 2-oxoglutarate dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis.

  De Carolis E., Chan F., Balsevich J., De Luca V.

  Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline bio ... >> More

  Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus. << Less

  Plant Physiol. 94:1323-1329(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.