Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis

Carolis, Emidio De; Chan, Francis Ka Ming; Balsevich, John; Luca, Vincenzo De

doi:10.1104/pp.94.3.1323

Cited by 82 publications

(57 citation statements)

References 26 publications

(29 reference statements)

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“…They are involved in a broad spectrum of primary and secondary biosynthetic pathways including the post-translational hydroxylation of proline and lysine residues in procollagens (Kivirikko et al, 1989), the biosynthesis of carnitine ) and blood-coagulation-factor VII (Stenflo et al, 1989) in mammals, the conversion of thymidine into uracil (Holme et al, 1970(Holme et al, , 1971Bankel et al, 1977) and of penicillins into cephalosporins (Baldwin and Abraham, 1988) in bacteria and fungi, the biosynthesis of clavulanic acid (Elson et al, 1987) and the macrolide antibioticum tylosin (Omura et al, 1984) in fungi, the formation of hydroxyproline-rich glycoproteins in plants (Chrispeels, 1969;Tanaka et al, 1980) and the biosynthesis of plant secondary products such a5 flavonoids Britsch et al, 1981), the alkaloids scopolamine (Hashimot0 and Yamada, 1986) and vindoline (De Carolis et al, 1990) and gibberellins (Hedden and Graebe, 1982).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of flayanone 3β‐hydroxylases

Britsch

Dedio²,

Saedler³

et al. 1993

European Journal of Biochemistry

101

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A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3P-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callisteplzus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from l ? hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53 % identity) of flavanone 3P-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(11) enzymes demonstrated the strict genetic conservation of 14 anuno acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of tlavanone 3P-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.Flavanone 3P-hydroxylase (FHT) catalyzes 3P-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols [( +)-dihydroflavonols] which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins in plants (Heller and Forkmann, 1988;Forkmann, 1991). The enzyme from Petunia hybrida has been purified to homogeneity and characterized as a typical 2-oxoglutarate-dependent dioxygenase (Britsch and Grisebach, 1986;Britsch, 1990). Recently, we have isolated a near-full-length FHTencoding cDNA from a Petunia library (Britsch et al., 1992). Heterologous expression of the cDNA in Escherichia coli led to the formation of highly active recombinant FHT, thereby verifying the identity of the cDNA clone.Antibodies against FHT from Petunia readily crossreact with FHT from a number of other sources and were used to characterize specific FHT mutants (Britsch, 1990; Koch, 1992). Therefore, structural similarities of the FHT protein from different plants can be assumed. A comparison of the deduced amino acid sequence of the Petunia FHT with the sequence of FHT from barley (Meldgaard, 1992) indeed showed a high degree of identity (75%; Britsch et al., 1992).Consequently, the FHT cDNA from Petunia should serve as a useful heterologous probe to clone this enzyme from other plant sources. Utilizing the 945-bp EcoRI fragment from Petunia FHT as a hybridization probe, we were able to clone the corresponding cDNAs from carnation, china aster and stock. In this study, we report the cloning and sequence analysis of these particular FHT enzymes, the analysis of the consensus sequence derived from six individual FHT sequences and the modification of purified recombinant enzyme with diethyl pyrocarbonate, performed to elucidate the possible function of strictly conserved histidines. Furthermore, the sequences were also compared with other known 2-oxoglutarate-dependent dioxygenases f...

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of flayanone 3β‐hydroxylases

Britsch

Dedio²,

Saedler³

et al. 1993

European Journal of Biochemistry

101

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“…Severa1 enzymes and intermediates in this pathway have been characterized. These enzymes include an O-methyltransferase (Fahn et al, 1985b), an N-methyltransferase , a 2-oxoglutarate-dependent dioxygenase (De Carolis et al, 1990), and an O-acetyltransferase (De Luca et al, 1985;Fahn et al, 1985a). Most of the intermediates required for these enzymatic steps have been identified in etiolated seedlings De Luca et al, 1986).…”

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confidence: 99%

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

1995

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Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catbarantbus roseus. Tabersonine 16-hydroxylase (1 6-OH) activity was detected in total protein extracts from young leaves of C. roseus using a nove1 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. l h e 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.

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Section: Introductionmentioning

confidence: 99%

“…The final reaction is catalyzed by acetyl coenzyme A (CoA):deacetylvindoline 4-O -acetyltransferase (DAT;De Luca et al, 1985;Power et al, 1990). Some of these enzymes are not expressed in cell cultures or in tissues unable to produce vindoline De Carolis et al, 1990).It has been suggested that certain tissues are required for alkaloid biosynthesis and/or accumulation. Yoder and Mahlberg (1976) used chemical indicators to identify laticifers and "specialized parenchyma cells" as the sites of alkaloid accumulation in C. roseus.…”

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confidence: 99%

“…The first pair of reactions are catalyzed by tabersonine 16-hydroxylase and S -adenosyl-L -methionine: 16-hydroxytabersonine-O -methyltransferase, respectively (StPierre and De Luca, 1995). The third to the last reaction is catalyzed by an S -adenosyl-L -methionine:2,3-dihydro-3-hydroxytabersonine-N -methyltransferase Dethier and De Luca, 1993), whereas 4-hydroxylation is catalyzed by desacetoxyvindoline 4-hydroxylase (D4H), a 2-oxoglutarate-dependent dioxygenase (De Carolis et al, 1990;De Carolis and De Luca, 1993). The final reaction is catalyzed by acetyl coenzyme A (CoA):deacetylvindoline 4-O -acetyltransferase (DAT;De Luca et al, 1985;Power et al, 1990).…”

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confidence: 99%

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Multicellular Compartmentation of Catharanthus roseus Alkaloid Biosynthesis Predicts Intercellular Translocation of a Pathway Intermediate

1999

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In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle ( Catharanthus roseus ). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O -acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed. INTRODUCTIONThe organs forming the plant body consist of several different cell types that are organized in relation to each other and that confer specific functions to the resulting organ. Each cell type emerges from an undifferentiated meristem according to a sophisticated and partially understood developmental program (Sylvester et al., 1996;von Arnim and Deng, 1996). The commitment to differentiate into specialized structures involves the perception by cells in the meristem of a complex array of signals, which communicate cellular age, position in relation to other cells, and hormonal balance. Environmental factors, such as light and temperature, also play a critical role in modulating these signals throughout the process of organogenesis (Bernier, 1988;Dale, 1988;Sylvester et al., 1996).In addition to morphogenesis, developmental processes result in biochemical specialization of cells for the biosynthesis and/or accumulation of secondary metabolites, such as phenylpropanoids (Ibrahim et al., 1987;Reinold and Hahlbrock, 1997), monoterpenoids (Fahn, 1988;McCaskill et al., 1992), and alkaloids (Robinson, 1974(Robinson, , 1981Nessler and Mahlberg, 1977;Eilert et al., 1985;Hashimoto and Yamada, 1994;Facchini and De Luca, 1995). Studies with germinating seedlings have suggested that alkaloid biosynthesis and accumulation are associated with seedling development (Weeks and Bush, 1974;De Luca et al., 1986;Aerts et al., 1994). Studies with mature plants also reveal this ty...

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Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis

Cited by 82 publications

References 26 publications

Molecular characterization of flayanone 3β‐hydroxylases

Molecular characterization of flayanone 3β‐hydroxylases

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

Multicellular Compartmentation of Catharanthus roseus Alkaloid Biosynthesis Predicts Intercellular Translocation of a Pathway Intermediate

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