RESUMEN
Higher plants produce a diverse array of secondary metabolites. These chemicals are synthesized from simple precursors through multistep reactions. To understand how plant cells developed such a complicated metabolism, we examined the plasticity of benzyl isoquinoline alkaloid biosynthesis in transgenic Eschscholzia californica cells with the ectopic expression of Coptis japonica scoulerine-9-O-methyltransferase (CjSMT). CjSMT catalyzes the O-methylation of scoulerine to produce tetrahydrocolumbamine (THC) in berberine biosynthesis and is not involved in benzophenanthridine alkaloid biosynthesis in E. californica. While a preliminary characterization confirmed that columbamine (oxidized product of THC) was produced in transgenic E. californica cells, many newly found peaks were not identified. Here, we report the identification of novel products, including allocryptopine and 10-hydroxychelerythrine. This result indicates that CjSMT reaction products were further converted by endogenous enzymes to produce double O-methylated compounds instead of a methylenedioxy ring at the 7,8-position of the original benzophenanthridine alkaloids. Further metabolite profiling revealed the enhanced diversification of the alkaloid profile in transgenic cells. Metabolic plasticity and the enzymes involved in metabolic diversity are discussed.
Asunto(s)
Alcaloides/biosíntesis , Bencilisoquinolinas/metabolismo , Eschscholzia/enzimología , Metaboloma , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Eschscholzia/genética , Espectrometría de Masas , Metiltransferasas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , ARN de Planta/genéticaRESUMEN
A metabolic pathway of 2,3,10,11-oxygenated tetrahydroprotoberberines having the OH group on ring D was demonstrated. Metabolism of (13)C- or D(2)-labeled precursors was studied in cell cultures of Macleaya, Corydalis, and Nandina species. The structures of alkaloid metabolites obtained from feeding experiments were determined by application of combined LC-NMR, LC-MS/MS, and LC-CD techniques. (S)-Tetrahydropseudoprotoberberine (5) was stereospecifically O-methylated to the S-isomer (12) in cell cultures of three plant species. This S-isomer was further N-methylated to the (S)-alpha-N-methyl salt (15), which was oxidized to produce the pseudoprotopine-type alkaloid (10) in cell cultures of Macleaya and Corydalis species. These transformations were the same as those of 2,3,9,10-oxygenated protoberberines. The tetrahydropseudoprotoberberines (5, 6, and 12) were dehydrogenated to pseudoprotoberberines (13, 16, and 14), respectively. Both the R- and S-enantiomers of 5 were dehydrogenated in Macleaya cordata different from the case of 2,3,9,10-oxygenated protoberberines. Precursor 7, with OH groups at C-10 and C-11, was O-methylated at C-10 in M. cordata and C. ochotensis var. raddeana, which was distinct from O-methylation in N. domestica, in which 7 was O-methylated at both C-11 and C-10. Stereoselective O-demethylation [(S)-5 to (S)-18] occurred in N. domestica.
Asunto(s)
Berberidaceae/química , Alcaloides de Berberina/metabolismo , Fumariaceae/química , Papaveraceae/química , Annonaceae/química , Alcaloides de Berberina/química , Biotransformación , Técnicas de Cultivo de Célula , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
Effective enantiomeric separations of 1-benzyl-N-methyltetrahydroisoquinolines were achieved using commercially available Chiralcel OD-H and OJ-H columns. Online LC-CD analysis allowed for the establishment of a correlation between the absolute configuration of the separated enantiomers and their characteristic CD transitions. LC-MS combined with LC-CD analysis permitted chiral purity determinations of O-methylated metabolites of nine phenolic 1-benzyl-N-methyltetrahydroisoquinolines in cell cultures of Corydalis, Macleaya, and Nandina species.
Asunto(s)
Hidrógeno/química , Quinolinas/química , Quinolinas/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Espectrometría de Masas , Metilación , Estructura Molecular , Sistemas en Línea , Extractos Vegetales/química , EstereoisomerismoRESUMEN
The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were identified from N. domestica. O-Methylpallidine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were also characterized for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were identified in the callus of N. domestica.
Asunto(s)
Alcaloides/química , Berberidaceae/metabolismo , Cromatografía Liquida/métodos , Dicroismo Circular/métodos , Espectroscopía de Resonancia Magnética/métodos , Extractos Vegetales/química , Espectrometría de Masas en Tándem/métodos , Berberidaceae/citología , Células Cultivadas , Estructura Molecular , Espectrofotometría UltravioletaRESUMEN
(S)-stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.
Asunto(s)
Alcaloides de Berberina/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Eschscholzia/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Acetatos/farmacología , Secuencia de Aminoácidos , Berberina/química , Berberina/metabolismo , Clonación Molecular , Ciclopentanos/farmacología , ADN Complementario/metabolismo , Eschscholzia/genética , Eschscholzia/metabolismo , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Oxilipinas , Reguladores del Crecimiento de las Plantas/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Semillas/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
From the secondary and tertiary alkaloidal fractions of the root and the bark of Xylopia parviflora (Annonaceae), the isoquinoline alkaloids, 10,11-dihydroxy-1,2-dimethoxynoraporphine and parvinine were isolated, along with 39 known alkaloids. Their structures were determined on the basis of analysis of spectroscopic data.
Asunto(s)
Alcaloides/química , Annonaceae/química , Isoquinolinas/química , Alcaloides/aislamiento & purificación , Isoquinolinas/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales/químicaRESUMEN
Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of beta-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 x 10(3) mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than alpha-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC50 4.5-5.8 microM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.
Asunto(s)
Anticarcinógenos/farmacología , Alcaloides de Berberina/farmacología , Isoquinolinas/farmacología , Anticarcinógenos/síntesis química , Anticarcinógenos/química , Antígenos Virales/efectos de los fármacos , Antígenos Virales/metabolismo , Alcaloides de Berberina/síntesis química , Alcaloides de Berberina/química , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimioprevención , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Radicales Libres/química , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/metabolismo , Humanos , Isoquinolinas/síntesis química , Isoquinolinas/química , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
The metabolism of 2,3,10,11-oxygenated protoberberine alkaloids was studied in cell cultures of Corydalis species. Without prior isolation, the structures of the metabolites were determined by LC-MS and LC-NMR analyses. Tetrahydropseudocoptisine alpha-N-metho salt, pseudoprotopine, and pseudomuramine were identified for the first time, and preliminary evidence for metabolic pathways to the formation of these alkaloids were obtained.
Asunto(s)
Alcaloides de Berberina/metabolismo , Corydalis/metabolismo , Alcaloides de Berberina/química , Biotransformación , Células Cultivadas , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Oxidación-ReducciónRESUMEN
From the quaternary alkaloidal fraction of the bark and the root of Xylopia parviflora (Annonaceae), four isoquinoline alkaloids, xylopinidine, dehydrocoreximine, N, N-dimethylanomurine and N-methylphoebine were isolated along with the known compounds, pycnarrhine, lotusine, 6,7-dimethoxy-2-methyl-isoquinolinium salt, 1,2-dehydroreticuline, (-)-phellodendrine, (+)-tembetarine, (-)-litcubine, (+)-magnoflorine, tetradehydroreticuline, (-)-oblongine, (+)-menisperine, (+)-N-methylcorydine, stepharanine, (+)-xanthoplanine, dehydrodiscretine, jatrorrhizine and palmatine. 3,4-Dihydro-6,7-dimethoxy-2-methyl-isoquinolinium and N-methylpurpuerine were isolated as natural products for the first time. Their structures were determined on the basis of spectroscopic evidence.
Asunto(s)
Alcaloides/química , Annonaceae/química , Isoquinolinas/química , Alcaloides/aislamiento & purificación , Isoquinolinas/aislamiento & purificación , Espectrometría de Masas , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Corteza de la Planta/química , Raíces de Plantas/química , Espectrofotometría UltravioletaRESUMEN
R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl [R-(-)-BPAP] is one of "catecholaminergic and serotonergic enhancers", which were proposed to improve symptoms through increase in impulse-evoked release of monoamine neurotransmitters for Parkinson's disease. It was reported that (-)-BPAP up-regulated the synthesis of neurotrophic factors in mouse astrocytes, suggesting the neuroprotective potency of (-)-BPAP. In this paper, the neuroprotective function of (-)-BPAP and the related compounds was examined against apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], a possible pathogenic toxin in Parkinson's disease, in human dopaminergic neuroblastoma SH-SY5Y cells. The anti-apoptotic activity was confirmed with some of (-)-BPAP analogues, and the mechanism was found to be due to the direct stabilization of mitochondrial membrane potential and the induction of anti-apoptotic Bcl-2. The studies on structure-activity relationship demonstrated that the potency to stabilize the mitochondrial membrane potential depended on the absolute stereo-chemical structure of BPAP derivatives. The compounds with dextrorotation prevented the mitochondrial permeability transition, whereas those with levorotation did not. The presence of a propargyl or propyl group at the amino residue of R-(-)-1-(benzofuran-2-yl)-2-propylamine increased potency to stabilize the membrane potential and prevent apoptosis. R-FPFS-1169 and R-FPFS-1180 had more potent to induce Bcl-2 and prevent apoptosis than the corresponding S-enantiomers. These results are discussed with the possible application of BPAP derivatives as neuroprotective agents in Parkinson's disease and other neurodegenerative disorders.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofuranos/farmacología , Fármacos Neuroprotectores/farmacología , Neurotoxinas/toxicidad , Alcaloides de Salsolina/toxicidad , Tetrahidroisoquinolinas/toxicidad , Benzofuranos/química , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fármacos Neuroprotectores/química , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores Dopaminérgicos/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction.
Asunto(s)
Benzofenantridinas/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Eschscholzia/metabolismo , Secuencia de Aminoácidos , Benzofenantridinas/química , Clonación Molecular , Eschscholzia/química , Eschscholzia/citología , Isoquinolinas/química , Datos de Secuencia Molecular , Estructura Molecular , Alineación de Secuencia , Especificidad por SustratoRESUMEN
In the present study, we attempted to elucidate the antinociceptive activity of Xylopia parviflora bark using the acetic acid-induced writhing test, hot plate test, and formalin test in mice. The MeOH extract (100 and 200 mg/kg, administered intraperitoneally (i.p.)) had an antinociceptive effect demonstrated by its inhibitory effects on writhing number induced by acetic acid. Three alkaloidal fractions exhibited significant antinociceptive effects in three animal models; the chloroform-soluble fraction, including secondary and tertiary alkaloids, exhibited the strongest effect. This result supported its use in folk medicine as an analgesic agent. We tested the main alkaloids of these fractions for their antinociceptive effects to clarify the active components. (+)-Corytuberine (6.3 and 12.5 mg/kg, i.p.) showed very strong activity, had a significant antinociceptive effect in the acetic acid-induced writhing test (with 49.4 and 98.9% reduction of writhes), in the hot plate test, and in the formalin test (with 55.4 and 90.6% inhibition during the first phase, and 73.9 and 99.9% during the second phase, respectively). (+)-Glaucine (12.5 and 25 mg/kg, i.p.) showed strong activity in three animal models, too. The activity of these compounds was also observed following oral administration in the acetic acid-induced writhing test.
Asunto(s)
Alcaloides/farmacología , Analgésicos/farmacología , Extractos Vegetales/farmacología , Xylopia/química , Alcaloides/administración & dosificación , Alcaloides/aislamiento & purificación , Analgésicos/administración & dosificación , Analgésicos/aislamiento & purificación , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Medicinas Tradicionales Africanas , Ratones , Dolor/tratamiento farmacológico , Dimensión del Dolor , Corteza de la Planta , Extractos Vegetales/administración & dosificaciónRESUMEN
Eschscholzia californica produces various types of isoquinoline alkaloids. The structural diversity of these chemicals is often due to cytochrome P450 (P450) activities. Members of the CYP719A subfamily, which are found only in isoquinoline alkaloid-producing plant species, catalyze methylenedioxy bridge-forming reactions. In this study, we isolated four kinds of CYP719A genes from E. californica to characterize their functions. These four cDNAs encoded amino acid sequences that were highly homologous to Coptis japonica CYP719A1 and E. californica CYP719A2 and CYP719A3, which suggested that these gene products may be involved in isoquinoline alkaloid biosynthesis in E. californica, especially in methylenedioxy bridge-forming reactions. Expression analysis of these genes showed that two genes (CYP719A9 and CYP719A11) were preferentially expressed in plant leaf, where pavine-type alkaloids accumulate, whereas the other two showed higher expression in root than in other tissues. They were suggested to have distinct physiological functions in isoquinoline alkaloid biosynthesis. Enzyme assay analysis using recombinant proteins expressed in yeast showed that CYP719A5 had cheilanthifoline synthase activity, which was expected based on the similarity of its primary structure to that of Argemone mexicana cheilanthifoline synthase (deposited at DDBJ/GenBanktrade mark/EMBL). In addition, enzyme assay analysis of recombinant CYP719A9 suggested that it has methylenedioxy bridge-forming activity toward (R,S)-reticuline. CYP719A9 might be involved in the biosynthesis of pavine- and/or simple benzylisoquinoline-type alkaloids, which have a methylenedioxy bridge in an isoquinoline ring, in E. californica leaf.
Asunto(s)
Alcaloides/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Eschscholzia/enzimología , Isoquinolinas/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , ADN Complementario/genética , Eschscholzia/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Datos de Secuencia Molecular , Estructura Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , ARN de Planta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.
Asunto(s)
Aporfinas/metabolismo , Bencilisoquinolinas/metabolismo , Coptis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Fenol/metabolismo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Aporfinas/química , Bencilisoquinolinas/química , Berberis/enzimología , Berberis/genética , Clonación Molecular , Coptis/citología , Coptis/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Datos de Secuencia Molecular , NADP/genética , NADP/metabolismo , Oxígeno/metabolismo , Fenol/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 mug/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.
Asunto(s)
Alcaloides/biosíntesis , Técnicas de Cultivo de Célula , Eschscholzia/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Interferencia de ARN , Alcaloides/análisis , Alcaloides/genética , Bencilisoquinolinas/análisis , Células Cultivadas , Regulación hacia Abajo , Eschscholzia/citología , Eschscholzia/genética , Vectores Genéticos/genética , Oxidorreductasas N-Desmetilantes/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Estereoisomerismo , Transcripción Genética/genéticaRESUMEN
(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Coptis/citología , Alcaloides/biosíntesis , Sitios de Unión , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/aislamiento & purificación , Línea Celular , Coptis/enzimología , ADN Complementario , ADN de Plantas , Dopamina/metabolismo , Hierro/química , Proteínas de Plantas , Análisis de Secuencia de ADN , TetrahidroisoquinolinasRESUMEN
(+/-)-1-Benzyl- N-methyltetrahydroisoquinolines 7-10 and 11-14 with one and two hydroxy groups on the aromatic rings, respectively, were fed individually to cultured cells of Corydalis and Macleaya species, respectively. The structures of the metabolites were determined by using combinatorial techniques, including LC/NMR, LC/MS-MS, and LC/CD. The enantiomeric excesses of the metabolites were derived from LC/CD and LC/MS-MS analyses. In cell cultures of Corydalis and Macleaya species, laudanine (7), with a hydroxy group at C-3', can form the berberine bridge at C-2' and C-6' to produce S- and R-enantiomers of 2,3,9,10- and 2,3,10,11-oxygenated protoberberines (20 and 21), respectively, whereas reticuline (11) and protosinomenine (12), incoporating a hydroxy group at C-3', form the berberine bridge at C-2' to furnish the S-enantiomer of 2,3,9,10-oxygenated protoberberines (23 and 21), respectively.
Asunto(s)
Corydalis/química , Fenoles/metabolismo , Tetrahidroisoquinolinas/metabolismo , Biotransformación , Células Cultivadas , Japón , Espectroscopía de Resonancia Magnética , Estructura Molecular , Fenoles/química , Estereoisomerismo , Relación Estructura-Actividad , Tetrahidroisoquinolinas/químicaRESUMEN
The metabolism of the phenolic 1-benzyltetrahydroisoquinoline alkaloids was studied in cell cultures of Macleaya and Corydalis species. The crude alkaloid fraction obtained from feeding experiments was investigated by application of the combined LC/NMR and LC/APCI-MS (/MS) techniques. Several metabolites were detected, and their structures (6 and 8-14) were identified. Bioconversion of the phenolic 1-benzyltetrahydroisoquinoline (2) into the pseudoprotoberberine (8) was demonstrated for the first time. LC/APCI-MS and LC/CD experiments carried out on a chiral column permitted the deduction of the major enantiomeric form of the chiral metabolites. Thus, the combination of NMR, MS, and CD data permitted the structural elucidation and stereochemical analysis of the metabolites in the extract matrix solution, without isolation and sample purification prior to the coupling experiments.