Vindoline pathway, biosynthesis

Murata J, de Luca V. Localization of tabersonine 16-hydroxylase and 16-OH tabersonine 16-O-methyl transferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Cafiiaranthus roseus. Plant J. 2005 44 581-594. 

In the plant, the enzymes of the vindoline pathway, DAT (236,247) and NMT (242), are only found in the aerial parts, and in particular the leaves, the site of vindoline biosynthesis. In C. roseus cell cultures these enzymes cannot be detected, either after elicitation or in photoautotrophic or hormone autotrophic cultures (242). However, two enzymes of the early steps of the vindoline pathway, TllH and 11-O-methyltransferase are also present in cell cultures although at lower levels than in the plant (241). In the plant, the highest TllH levels are found in young leaves, whereas it is absent in stems and old leaves. 

Terpenoid indole alkaloid biosynthetic enzymes are associated with at least three different cell types in C. roseus TDC and STR are localized to the epidermis of aerial organs and the apical meristem of roots, D4H and DAT are restricted to the laticifers and idio-blasts of leaves and stems, and GlOH is found in internal parenchyma of aerial organs (St-Pierre et al. 1999 Buriat et al. 2004) thus, vindoline pathway intermediates must be translocated between cell types. Moreover, enzymes involved in terpenoid indole alkaloid biosynthesis in C. roseus are also localized to at least five subcellular compartments TDC, D4H and DAT are in the cytosol, STR and the peroxidase that couples catharanthine to vinblastine are localized to the vacuole indicating transport of tryptamine across the tono-plast, SGD is a soluble enzyme associated with the cytoplasmic face of the endoplasmic reticulum, the P450-dependent monooxygenases are integral endomembrane proteins, and the N-methyltransferase involved in vindoline biosynthesis is localized to thylakoid membranes (De Luca and St-Pierre 2000). 

Salutaridinol 7-0-acetyltransferase catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-Oacetate, the immediate precursor of thebaine along the morphine biosynthetic pathway in P. somniferum (Fig. 10.7).26 Acetyl CoA-dependent acetyltransferases have an important role in plant alkaloid metabolism. They are involved in the synthesis of monoterpenoid indole alkaloids in medicinal plant species such as Rauwolfia serpentina. In this plant, the enzyme vinorine synthase transfers an acetyl group from acetyl CoA to 16-epi-vellosimine to form vinorine. This acetyl transfer is accompanied by a concomitant skeletal rearrangement from the sarpagan- to the ajmalan-type (reviewed in2). An acetyl CoA-dependent acetyltransferase also participates in vindoline biosynthesis in Catharanthus roseus, the source of the chemotherapeutic dimeric indole alkaloid vinblastine (reviewed in2). Acetyl CoA deacetylvindoline 4-O-acetyltransferase catalyzes the last step in vindoline biosynthesis. A cDNA encoding acetyl CoA deacetylvindoline 4-0-acetyltransferase was recently successfully isolated.27... 

The translocation of pathway intermediates is also suggested by differential localization of TLA biosynthetic enzymes. In situ hybridization and immunolocalization studies have shown that CYP72A1, TDC, and STR are localized to the epidermis of immature leaves, stems, and flower buds.52,147 In contrast, D4H and DAT are associated with laticifers and idioblasts of shoot organs. Laticifers and idioblasts are distributed throughout the mesophyll in C. roseus leaves, and are often several cell layers away from the epidermis. Vindoline biosynthesis involves at least two distinct cell types and requires the intercellular translocation of a pathway intermediate (Fig.7.9B). The differential localization of the early and late steps of vindoline biosynthesis might partially explain why this alkaloid is not produced in dedifferentiated C. roseus cell cultures. 

DE LUCA, V., BALSEVICH, J TYLER, R.T., EILERT, U PANCHUK, B.D., KURZ, W.G.W., Biosynthesis of indole alkaloids Developmental regulation of the biosynthetic pathway from tabersonine to vindoline in Catharanthus roseus. J. Plant Physiol., 1986,125, 147-156. 

A large amount of structural information is available that describes the variety of indole alkaloids produced in plants. This has recently been followed by significant increases in our knowledge of the biosynthetic pathways that lead to their production and of the genes involved. Several reviews have appeared recently that describe the chemistry, biochemistry, cell and molecular biology of alkaloid biosynthesis.1 3 This chapter will selectively review recently characterized genes that appear to be responsible for the diversity and complexity of monoterpenoid indole alkaloids produced by plants. A particular focus will be on the reactions leading to the biosynthesis of vindoline (Fig. 8.1) in Catharanthus roseus. 

Fig. 8.1 Sequence of reactions and pathways involved in the biosynthesis of indole alkaloids in Catharanthus roseus. The dotted lines indicate multiple and/or uncharacterized enzyme steps. Tryptophan decarboxylase (TDC), Geraniol Hydroxylase (GH), Deoxyloganin synthase (DS), Secologanin Synthase (SLS) Strictosidine synthase (STR1), Strictosidine glucosidase (SG), Tabersonine-16-hydroxylase (T16H), Tabersonine 6,7-eposidase (T6,7E), Desacetoxyvindoline-4-hydroxylase (D4H), Deacetyl-vindoline-4-O-acetyltransferase (DAT) and Minovincinine-19-O-acetyltransferase (MAT) represent some of the enzyme steps that have been characterized.

The association of cytochrome P450s with organelles other than the ER may have important implications for the biosynthesis of alkaloids. It may help to explain why biochemical localization studies have shown that certain reactions in a pathway occur within the chloroplast. In the case of Catharanthus alkaloids, the 3 rd to last step in vindoline biosynthesis involves a chloroplast thylakoid associated N-methyltransferase.22 The arguments for participation of chloroplasts in this reaction include the possibility that the previous step involving hydration of the 2,3 double bond might require a chloroplast based oxidation reaction (Fig 8.6). The conclusive identification of specific cytochrome P4S0 enzymes in chloroplasts suggests that this hypothesis should be tested. 

Terpenoid indole alkaloid biosynthesis actually starts with the coupling of tryptamine and secologanin (Fig. 12). In the next step, a glucosidase splits off the sugar moiety and the reactive dialdehyde formed is further converted through different pathways to a cascade of products, including ajmalicine, catharanthine, tabersonine, and vindoline. 

While the biosynthesis of catharanthine and tabersonine is poorly characterized (Scheme 4.5), the biosynthetic pathway from tabersonine to vindoline has been extensively studied [2]. Thus, the hydroxylation of tabersonine at position 16 represents the first reaction leading to vindoline (Scheme 4.6), and it is catalyzed by tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme... 

The most important, indeed crucial, idea put forward on the biosynthesis of these alkaloids was that they are formed by fragmentation of a cyclopentane monoterpene [as (6.237)]. The pathway outlined in Scheme 6.43 indicates how the major skeletal types represented by ajmalicine (6.243) and akuammicine (6.242), catharan-thine (6.239), and vindoline (6.244) may be formed where one of... 

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