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1.
Plant Physiol ; 187(2): 846-857, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34608956

RESUMEN

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the ß-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.


Asunto(s)
Catharanthus/genética , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Catharanthus/enzimología , Catharanthus/metabolismo , Silenciador del Gen , Genes de Plantas , Proteínas de Plantas/metabolismo , Alcaloides de Triptamina Secologanina/metabolismo , Transducción de Señal
2.
Sci Rep ; 8(1): 15059, 2018 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-30305670

RESUMEN

Catharanthus roseus is a commercial source for anti-cancer terpenoid indole alkaloids (TIAs: vincristine and vinblastine). Inherent levels of these TIAs are very low, hence research studies need to focus on enhancing their levels in planta. Since primary metabolism provides precursors for specialized-metabolism, elevating the former can achieve higher amounts of the latter. Cell Wall Invertase (CWIN), a key enzyme in sucrose-metabolism catalyses the breakdown of sucrose into glucose and fructose, which serve as carbon-skeleton for specialized-metabolites. Understanding CWIN regulation could unravel metabolic-engineering approaches towards enhancing the levels of TIAs in planta. Our study is the first to characterize CWIN at gene-expression level in the medicinal plant, C. roseus. The CWINs and their inter-relationship with sucrose and TIA metabolism was studied at gene and metabolite levels. It was found that sucrose-supplementation to C. roseus leaves significantly elevated the monomeric TIAs (vindoline, catharanthine) and their corresponding genes. This was further confirmed in cross-species, wherein Nicotiana benthamiana leaves transiently-overexpressing CrCWIN2 showed significant upregulation of specialized-metabolism genes: NbPAL2, Nb4CL, NbCHS, NbF3H, NbANS, NbHCT and NbG10H. The specialized metabolites- cinnamic acid, coumarin, and fisetin were significantly upregulated. Thus, the present study provides a valuable insight into metabolic-engineering approaches towards augmenting the levels of therapeutic TIAs.


Asunto(s)
Catharanthus/enzimología , Catharanthus/metabolismo , Pared Celular/enzimología , Estrés Fisiológico , beta-Fructofuranosidasa/genética , Antioxidantes/metabolismo , Catharanthus/citología , Catharanthus/genética , Simulación por Computador , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Metaboloma , Especificidad de Órganos/genética , Filogenia , Hojas de la Planta/metabolismo , Solubilidad , Estrés Fisiológico/genética , Nicotiana , beta-Fructofuranosidasa/metabolismo
3.
Biotechnol Bioeng ; 115(3): 673-683, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29105731

RESUMEN

The monoterpene indole alkaloids vindoline and catharanthine, which are exclusively synthesized in the medicinal plant Catharanthus roseus, are the two important precursors for the production of pharmaceutically important anti-cancer medicines vinblastine and vincristine. Hairy root culture is an ideal platform for alkaloids production due to its industrial scalability, genetic and chemical stability, and availability of genetic engineering tools. However, C. roseus hairy roots do not produce vindoline due to the lack of expression of the seven-step pathway from tabersonine to vindoline [Murata & De Luca (2015) Plant Journal, 44, 581-594]. The present study describes the genetic engineering of the first two genes tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT) in the missing vindoline pathway under the control of a glucocorticoid-inducible promoter to direct tabersonine toward vindoline biosynthesis in C. roseus hairy roots. In two transgenic hairy roots, the induced overexpression of T16H and 16OMT resulted in the accumulation of vindoline pathway metabolites 16-hydroxytabersonine and 16-methoxytabersonine. The levels of root-specific alkaloids, including lochnericine, 19-hydroxytabersonine and hörhammericine, significantly decreased in the induced hairy roots in comparison to the uninduced control lines. This suggests tabersonine was successfully channeled to the vindoline pathway away from the roots competing pathway based on the overexpression. Interestingly, another two new metabolites were detected in the induced hairy roots and proposed to be the epoxidized-16-hydroxytabersonine and lochnerinine. Thus, the introduction of vindoline pathway genes in hairy roots can cause unexpected terpenoid indole alkaloids (TIA) profile alterations. Furthermore, we observed complex transcriptional changes in TIA genes and regulators detected by RT-qPCR which highlight the tight regulation of the TIA pathway in response to T16H and 16OMT engineering in C. roseus hairy roots.


Asunto(s)
Catharanthus/enzimología , Sistema Enzimático del Citocromo P-450/biosíntesis , Expresión Génica , Alcaloides Indólicos/metabolismo , Proteínas de Plantas/biosíntesis , Raíces de Plantas/enzimología , Plantas Modificadas Genéticamente/enzimología , Quinolinas/metabolismo , Catharanthus/genética , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Plantas/genética , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/genética
4.
J Biol Chem ; 292(35): 14659-14667, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28701463

RESUMEN

The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon (Antirrhinum majus, Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction-cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control.


Asunto(s)
Transferasas Alquil y Aril/metabolismo , Antirrhinum/enzimología , Iridoides/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Monoterpenos Acíclicos , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Biocatálisis , Dominio Catalítico , Catharanthus/enzimología , Iridoides/química , Estructura Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Mutación , NADP/química , NADP/metabolismo , Oxidación-Reducción , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Homología Estructural de Proteína , Especificidad por Sustrato , Terpenos/química , Terpenos/metabolismo
5.
Nature ; 492(7427): 138-42, 2012 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-23172143

RESUMEN

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.


Asunto(s)
Biocatálisis , Catharanthus/enzimología , Iridoides/química , Iridoides/metabolismo , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Catharanthus/genética , Catharanthus/metabolismo , Ciclización , Reacción de Cicloadición , Datos de Secuencia Molecular , Monoterpenos/metabolismo , NADP/metabolismo , Oxidorreductasas/metabolismo , Extractos Vegetales/química , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Especificidad por Sustrato
6.
Mol Biol Rep ; 39(3): 3235-43, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21706164

RESUMEN

The enzyme geranylgeranyl diphosphate synthase (GGPS: EC 2.5.1.1, EC 2.5.1.10, EC 2.5.1.29) catalyses the formation of geranylgeranyl diphosphate (GGPP) from isopentenyl diphosphate and dimethylallyl diphosphate via three successive condensation reactions. A full-length nucleotide sequence of GGPS (named CrGGPS) was cloned from the medicinal plant Catharanthus roseus. The deduced polypeptide has 383 amino acids with a calculated mass of 41.6 kDa and possesses prenyltransferase signatures characteristic of plant type II GGPS. The enzyme was characterized by functional complementation in carotenoid accumulating strains of Escherichia coli. When cultures of Catharanthus cell lines were treated with methyljasmonate, no specific increase in transcript levels were observed. In plants, GGPS are encoded by a small multigene family and the isoforms have been shown to be localized in three different subcellular compartments: chloroplast, endoplasmic reticulum and mitochondria. We investigated the subcellular distribution of CrGGPS through transient transformations of C. roseus cells with a yellow fluorescent protein-fused construct. Our results clearly indicate that CrGGPS is located to plastids within stroma and stromules.


Asunto(s)
Catharanthus/enzimología , Farnesiltransferasa/genética , Acetatos , Secuencia de Aminoácidos , Proteínas Bacterianas , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Ciclopentanos , Cartilla de ADN/genética , ADN Complementario/biosíntesis , Escherichia coli , Farnesiltransferasa/metabolismo , Prueba de Complementación Genética , Espacio Intracelular/metabolismo , Proteínas Luminiscentes , Microscopía Fluorescente , Datos de Secuencia Molecular , Oxilipinas , Plastidios/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN
7.
J Exp Bot ; 62(8): 2841-54, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21357771

RESUMEN

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.


Asunto(s)
Catharanthus/enzimología , Peróxido de Hidrógeno/metabolismo , Peroxidasa/metabolismo , Fenoles/metabolismo , Plantas Medicinales/enzimología , Vacuolas/enzimología , Ácido Ascórbico/metabolismo , Catharanthus/efectos de la radiación , Catharanthus/ultraestructura , Isoenzimas/metabolismo , Luz , Espectrometría de Masas , Células del Mesófilo/citología , Células del Mesófilo/enzimología , Células del Mesófilo/efectos de la radiación , Células del Mesófilo/ultraestructura , Oxidación-Reducción/efectos de la radiación , Fenoles/química , Fenoles/aislamiento & purificación , Extractos Vegetales , Hojas de la Planta/enzimología , Hojas de la Planta/efectos de la radiación , Hojas de la Planta/ultraestructura , Plantas Medicinales/efectos de la radiación , Plantas Medicinales/ultraestructura , Protoplastos/metabolismo , Espectrofotometría Ultravioleta , Especificidad por Sustrato/efectos de la radiación , Factores de Tiempo , Vacuolas/efectos de la radiación , Vacuolas/ultraestructura
9.
Nature ; 468(7322): 461-4, 2010 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-21048708

RESUMEN

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.


Asunto(s)
Carbono/metabolismo , Catharanthus/metabolismo , Cloro/metabolismo , Plantas Medicinales/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos/biosíntesis , Productos Biológicos/genética , Biotecnología/métodos , Carbono/química , Catharanthus/enzimología , Catharanthus/genética , Cloro/química , Halogenación , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Alcaloides Indólicos/metabolismo , Monoterpenos/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Rhizobium/genética , Alcaloides de Triptamina Secologanina/metabolismo , Biología Sintética/métodos , Técnicas de Cultivo de Tejidos , Transgenes , Triptófano/metabolismo
10.
Colloids Surf B Biointerfaces ; 59(2): 150-7, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17560094

RESUMEN

Catharanthus roseus (L.) G. Don plants were grown in different water regimes in order to study the drought induced osmotic stress and proline (PRO) metabolism, antioxidative enzyme activities and indole alkaloid accumulation. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress from 30 days after sowing (DAS) and regular irrigation was kept as control. The plants were uprooted on 41DAS (10DID), 46DAS (15DID) and 51DAS (20DID). The drought stressed plants showed increased aminoacid (AA), glycine betaine (GB) and PRO contents and decreased proline oxidase (PROX) and increased gamma-glutamyl kinase (gamma-GK) activities when compared to control. The antioxidative enzymes like peroxidase (POX) and polyphenol oxidase (PPO) increased to a significant level in drought stressed plants when compared to control. The drought stressed C. roseus plants showed an increase in total indole alkaloid content in shoots and roots when compared to well-watered control plants. Our results suggest that the cultivation of medicinal plants like C. roseus in water deficit areas would increase its PRO metabolism, osmoregulation, defense system and the level of active principles.


Asunto(s)
Antioxidantes/metabolismo , Catharanthus/enzimología , Alcaloides Indólicos/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Catharanthus/metabolismo , Deshidratación/metabolismo , Ósmosis/fisiología , Agua/metabolismo
12.
FEBS Lett ; 580(18): 4501-7, 2006 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-16870181

RESUMEN

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its importance, no expressed sequence tag (EST) analysis of this plant has been reported. Two cDNA libraries were generated from RNA isolated from the base part of young leaves and from root tips to select 9,824 random clones for unidirectional sequencing, to yield 3,327 related sequences and 1,696 singletons by cluster analysis. Putative functions of 3,663 clones were assigned, from 5,023 non-redundant ESTs to establish a resource for transcriptome analysis and gene discovery in this medicinal plant.


Asunto(s)
Catharanthus/genética , Etiquetas de Secuencia Expresada , Transportadoras de Casetes de Unión a ATP/genética , Catharanthus/enzimología , Catharanthus/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Etiquetas de Secuencia Expresada/química , Biblioteca de Genes , Genes de Plantas , Glucosiltransferasas/genética , Alcaloides Indólicos/metabolismo , Metiltransferasas/genética , Microdisección/métodos , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética
13.
Planta Med ; 71(6): 572-4, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15971133

RESUMEN

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid indole alkaloids, some of which have high therapeutic interest. The biotechnological approach with cells in vitro remains an alternative to the field culture of periwinkle for the production of such compounds. We previously reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the accumulation of alkaloids in periwinkle cell suspensions. In this work, we investigated the effects of these hormones on the regulation of several genes of the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene greatly enhanced the expression of the geraniol 10-hydroxylase gene. When given together, these hormones also increased the expression of three genes belonging to the methyl-erythritol pathway. These results make it possible to consider elements of cytokinin and ethylene signalling pathways as tools for improving terpenoid indole alkaloid production through metabolic engineering.


Asunto(s)
Catharanthus/metabolismo , Alcaloides Indólicos/metabolismo , Fitoterapia , Terpenos/metabolismo , Catharanthus/enzimología , Catharanthus/genética , Técnicas de Cultivo , Citocininas , Etilenos , Regulación de la Expresión Génica de las Plantas , Humanos , Alcaloides Indólicos/química , Raíces de Plantas , Regiones Promotoras Genéticas , Ingeniería de Proteínas , Terpenos/química
14.
Phytochemistry ; 65(8): 1085-94, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15110688

RESUMEN

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.


Asunto(s)
Catharanthus/enzimología , Flavanonas/metabolismo , Flavonoides/metabolismo , Metiltransferasas/metabolismo , Oxigenasas/metabolismo , Secuencia de Aminoácidos , ADN Complementario/genética , Intrones/genética , Isoenzimas , Ácidos Cetoglutáricos/metabolismo , Metilación , Metiltransferasas/genética , Datos de Secuencia Molecular , Oxigenasas/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
15.
Protoplasma ; 222(1-2): 97-105, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-14513315

RESUMEN

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.


Asunto(s)
Catharanthus/enzimología , Peroxidasa/aislamiento & purificación , Peroxidasa/metabolismo , Vinblastina/análogos & derivados , Vinblastina/biosíntesis , Catharanthus/genética , Histocitoquímica , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Estructura Molecular , Oxidación-Reducción , Extractos Vegetales , Hojas de la Planta/química , Hojas de la Planta/citología , Especificidad por Sustrato , Vacuolas/metabolismo
16.
J Biotechnol ; 96(2): 193-203, 2002 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-12039535

RESUMEN

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.


Asunto(s)
Alcaloides/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/biosíntesis , Catharanthus/enzimología , Catharanthus/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/metabolismo , Catharanthus/citología , Catharanthus/genética , Células Cultivadas , Estructura Molecular , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Medicinales/citología , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Plantas Medicinales/metabolismo
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