RESUMEN
Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.
RESUMEN
The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.
Asunto(s)
Codeína/biosíntesis , Morfina/biosíntesis , Papaver/metabolismo , Hidrocodona/análogos & derivados , Hidrocodona/metabolismo , Isomerasas/fisiología , Opio/metabolismo , Papaver/enzimología , Tebaína/metabolismoRESUMEN
Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.
Asunto(s)
Bencilisoquinolinas/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Membrana Celular/metabolismo , Codeína/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Nucleobases/genéticaRESUMEN
The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Tebaína/metabolismo , Conformación Molecular , Proteínas de Saccharomyces cerevisiae/química , Tebaína/químicaRESUMEN
Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, ß-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.
Asunto(s)
Ephedra sinica/enzimología , Efedrina/metabolismo , Metiltransferasas/metabolismo , Seudoefedrina/metabolismo , Vías Biosintéticas , Ephedra sinica/genética , Ephedra sinica/metabolismo , Metiltransferasas/genética , Metabolismo Secundario , Especificidad por Sustrato , TranscriptomaRESUMEN
Since the 1930s, parabens have been employed widely as preservatives in food, pharmaceutical, and personal care products. These alkyl esters of benzoic acid occur naturally in a broad range of plant species, where they are thought to enhance overall fitness through disease resistance and allelopathy. Current manufacture of parabens relies on chemical synthesis and the processing of 4-hydroxybenzoate as a precursor. A variety of bio-based production platforms have targeted 4-hydroxybenzoate for a greener alternative to chemical manufacturing, but parabens have yet to be made in microbes. Here, we deploy the plant enzyme benzoic acid carboxyl methyltransferase together with four additional recombinant enzymes to produce methylparaben in Escherichia coli. The feasibility of a tyrosine-dependent route to methylparaben is explored, establishing a framework for linking paraben production to emerging high-tyrosine E. coli strains. However, our use of a unique plant enzyme for bio-based methylparaben biosynthesis is potentially applicable to any microbial system engineered for the manufacture of 4-hydroxybenzoate.
Asunto(s)
Escherichia coli/metabolismo , Parabenos/química , Estudios de Factibilidad , Microbiología IndustrialRESUMEN
Gene fusions have recently attracted attention especially in the field of plant specialized metabolism. The occurrence of a gene fusion, in which originally separate gene products are combined into a single polypeptide, often corresponds to the functional association of individual components within a single metabolic pathway. Examples include gene fusions implicated in benzylisoquinoline alkaloid (BIA), terpenoid, and amino acid biosynthetic pathways, in which distinct domains within a fusion catalyze consecutive, yet independent reactions. Both genomic and transcriptional mechanisms result in the fusion of gene products, which can include partial or complete domain repeats and extensive domain shuffling as evident in the BIA biosynthetic enzyme norcoclaurine synthase. Artificial gene fusions are commonly deployed in attempts to engineer new or improved pathways in plants or microorganisms, based on the premise that fusions are advantageous. However, a survey of functionally characterized fusions in microbial systems shows that the functional impact of fused gene products is not straightforward. For example, whereas enzyme fusions might facilitate the metabolic channeling of unstable intermediates, this channeling can also occur between tightly associated independent enzymes. The frequent occurrence of both fused and unfused enzymes in plant and microbial metabolism adds additional complexity, in terms of both pathway functionality and evolution.
Asunto(s)
Fusión Génica , Plantas/genética , Plantas/metabolismo , Aminoácidos/metabolismo , Bacterias/genética , Bacterias/metabolismo , BiotecnologíaRESUMEN
The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.
Asunto(s)
Aldehído Reductasa/metabolismo , Carbohidrato Epimerasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , Morfina/biosíntesis , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Aldehído Reductasa/genética , Aldo-Ceto Reductasas , Alcaloides/biosíntesis , Alcaloides/química , Secuencia de Bases , Bencilisoquinolinas/química , Bencilisoquinolinas/metabolismo , Bromoviridae/genética , Bromoviridae/metabolismo , Carbohidrato Epimerasas/antagonistas & inhibidores , Carbohidrato Epimerasas/genética , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Fusión Génica , Intrones , Ligasas/genética , Ligasas/metabolismo , Datos de Secuencia Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfina/química , Sistemas de Lectura Abierta , Opio/química , Opio/metabolismo , Oxidación-Reducción , Papaver/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , EstereoisomerismoRESUMEN
Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4'-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6' coupling and four O-methyl groups at C6, C7, C3', and C4' as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3' and 4' hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4'OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4'-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3'-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3'- and 7'-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.
Asunto(s)
Aporfinas/metabolismo , Metiltransferasas/metabolismo , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Bencilisoquinolinas/metabolismo , Alcaloides de Berberina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Isoquinolinas/metabolismo , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Papaveraceae/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/metabolismo , Plantas Medicinales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tetrahidropapaverolina/metabolismoRESUMEN
Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.
Asunto(s)
Morfina/biosíntesis , Papaver/citología , Células Vegetales/enzimología , Proteínas de Plantas/metabolismo , Alcaloides/metabolismo , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Morfinanos/metabolismo , Papaver/metabolismo , Proteómica , Tetrahidroisoquinolinas/metabolismo , Tebaína/metabolismoRESUMEN
Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using L-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
Asunto(s)
Ephedra sinica/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Transaminasas/química , Transaminasas/metabolismo , Biocatálisis , Estabilidad de Enzimas , Ephedra sinica/química , Ephedra sinica/genética , Ephedra sinica/metabolismo , Efedrina/metabolismo , Cinética , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Transaminasas/genética , Transaminasas/aislamiento & purificaciónRESUMEN
BACKGROUND: Recent progress toward the elucidation of benzylisoquinoline alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties. RESULTS: We applied a multi-platform approach incorporating four different analytical methods to examine 20 non-model, BIA-accumulating plant species. Plants representing four families in the Ranunculales were chosen based on reported BIA content, taxonomic distribution and importance in modern/traditional medicine. One-dimensional (1)H NMR-based profiling quantified 91 metabolites and revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content. Mono- and disaccharide sugars were generally lower in roots and rhizomes compared with stems, and a variety of metabolites distinguished callus tissue from intact plant organs. Direct flow infusion tandem mass spectrometry provided a broad survey of 110 lipid derivatives including phosphatidylcholines and acylcarnitines, and high-performance liquid chromatography coupled with UV detection quantified 15 phenolic compounds including flavonoids, benzoic acid derivatives and hydroxycinnamic acids. Ultra-performance liquid chromatography coupled with high-resolution Fourier transform mass spectrometry generated extensive mass lists for all species, which were mined for metabolites putatively corresponding to BIAs. Different alkaloids profiles, including both ubiquitous and potentially rare compounds, were observed. CONCLUSIONS: Extensive metabolite profiling combining multiple analytical platforms enabled a more complete picture of overall metabolism occurring in selected plant species. This study represents the first time a metabolomics approach has been applied to most of these species, despite their importance in modern and traditional medicine. Coupled with genomics data, these metabolomics resources serve as a key resource for the investigation of BIA biosynthesis in non-model plant species.
Asunto(s)
Alcaloides/metabolismo , Bencilisoquinolinas/metabolismo , Magnoliopsida/genética , Metaboloma , Proteínas de Plantas/genética , Berberidaceae/genética , Berberidaceae/metabolismo , Magnoliopsida/metabolismo , Menispermaceae/genética , Menispermaceae/metabolismo , Papaveraceae/genética , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Ranunculaceae/genética , Ranunculaceae/metabolismoRESUMEN
BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.
Asunto(s)
Alcaloides/metabolismo , Bencilisoquinolinas/metabolismo , Magnoliopsida/genética , Proteínas de Plantas/genética , Transcriptoma , Berberidaceae/genética , Berberidaceae/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Magnoliopsida/metabolismo , Menispermaceae/genética , Menispermaceae/metabolismo , Datos de Secuencia Molecular , Papaveraceae/genética , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Ranunculaceae/genética , Ranunculaceae/metabolismo , Análisis de Secuencia de ADNRESUMEN
The psychedelic prodrug psilocybin has shown therapeutic benefits for the treatment of numerous psychiatric conditions. Despite positive clinical end points targeting depression and anxiety, concerns regarding the duration of the psychedelic experience produced by psilocybin, associated with enduring systemic exposure to the active metabolite psilocin, pose a barrier to its therapeutic application. Our objective was to create a novel prodrug of psilocin with similar therapeutic benefits but a reduced duration of psychedelic effects compared with psilocybin. Here, we report the synthesis and functional screening of 28 new chemical entities. Our strategy was to introduce a diversity of cleavable groups at the 4-hydroxy position of the core indole moiety to modulate metabolic processing. We identified several novel prodrugs of psilocin with altered pharmacokinetic profiles and reduced pharmacological exposure compared with psilocybin. These candidate prodrugs have the potential to maintain the long-term benefits of psilocybin therapy while attenuating the duration of psychedelic effects.
Asunto(s)
Alucinógenos , Profármacos , Humanos , Psilocibina/farmacología , Psilocibina/uso terapéutico , Alucinógenos/farmacología , Alucinógenos/uso terapéutico , Profármacos/farmacología , Profármacos/uso terapéutico , Trastornos de Ansiedad/tratamiento farmacológicoRESUMEN
Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.
Asunto(s)
Benzofenantridinas/biosíntesis , Biocatálisis , Flavoproteínas/metabolismo , Opio/metabolismo , Oxidorreductasas/metabolismo , Papaver/enzimología , Papaverina/biosíntesis , Benzofenantridinas/química , Pruebas de Enzimas , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas/genética , Estudios de Asociación Genética , Isoquinolinas/química , Oxidorreductasas/genética , Papaver/genética , Papaverina/química , Filogenia , Virus de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites with a long history of investigation. Although the ecophysiological functions of most BIAs are unknown, the medicinal properties of many compounds have been exploited for centuries. These include the narcotic analgesics codeine and morphine, the antimicrobial agents sanguinarine and berberine, and the antitussive and anticancer drug noscapine. BIA biosynthesis involves a restricted number of enzyme types that catalyze landmark coupling reactions and subsequent functional group modifications. A pathogenesis-related (PR)10/Bet v1 'Pictet-Spenglerase', several O-methyl-, N-methyl- and O-acetyltransferases, cytochromes P450, FAD-dependent oxidases, non-heme dioxygenases and NADPH-dependent reductases have been implicated in the multistep pathways leading to structurally diverse alkaloids. A small number of plant species, including opium poppy (Papaver somniferum) and other members of the Ranunculales, have emerged as model systems to study BIA metabolism. The expansion of resources to include a wider range of plant species is creating an opportunity to investigate previously uncharacterized BIA pathways. Contemporary knowledge of BIA metabolism reflects over a century of research coupled with the development of key innovations such as radioactive tracing, enzyme isolation and molecular cloning, and functional genomics approaches such as virus-induced gene silencing. Recently, the emergence of transcriptomics, proteomics and metabolomics has expedited the discovery of new BIA biosynthetic genes. The growing repository of BIA biosynthetic genes is providing the parts required to apply emerging synthetic biology platforms to the development of production systems in microbes as an alternative to plants as a commecial source of valuable BIAs.
Asunto(s)
Alcaloides/metabolismo , Bencilisoquinolinas/metabolismo , Alcaloides/biosíntesis , Alcaloides/química , Alcaloides/farmacología , Bencilisoquinolinas/química , Bencilisoquinolinas/farmacología , Vías Biosintéticas , Genómica , Metabolómica , Biología SintéticaRESUMEN
Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3'-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT), which yields (S)-reticuline, and the coupled salutaridinol-7-O-acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert (S)-norcoclaurine to (S)-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.
Asunto(s)
Morfina , Papaver , Vías Biosintéticas , Retroalimentación , Morfina/metabolismo , Saccharomyces cerevisiae/metabolismo , Tebaína/metabolismoRESUMEN
Two previously undetected enzymes involved in morphine biosynthesis and unique among plants to opium poppy have been identified as non-heme dioxygenases, in contrast to the functionally analogous cytochrome P450s found in mammals. We used functional genomics to isolate thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), the only known 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze O-demethylation. Virus-induced gene silencing of T6ODM and CODM in opium poppy efficiently blocked metabolism at thebaine and codeine, respectively.
Asunto(s)
Dioxigenasas/metabolismo , Morfina/biosíntesis , Papaver/metabolismo , Biocatálisis , Metilación , Morfina/química , Papaver/química , Papaver/enzimologíaRESUMEN
Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.
Asunto(s)
Alcaloides , Bencilisoquinolinas , Papaver , Papaver/genética , Papaver/metabolismo , Látex/química , Alcaloides/química , Bencilisoquinolinas/metabolismo , Morfina , Saccharomyces cerevisiae/metabolismoRESUMEN
Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high-resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and pulsed Q collision-induced dissociation (PQD) MS2 fragmentation, with MS2 CID followed by MS3 and MS4 fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.