Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.     

CYP719B1 Is Salutaridine Synthase, the C-C Phenol-coupling Enzyme of Morphine Biosynthesis in Opium Poppy

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.The C-O or C-C phenol-couple is widely present in the plant kingdom in natural product biosynthetic processes such as alkaloid (1), lignan (2), lignin (3), and gallotannin (4) formation. Phenol-coupling reactions in nature were thought to be catalyzed by a variety of oxidative enzymes with broad substrate specificity such as peroxidases, polyphenol oxidases, and laccases. More recently, several enzymes discovered to be responsible for the formation of intermolecular C-O phenol and intramolecular C-C phenol-couples were found to be highly regio- and/or stereoselective catalysts. The first intermolecular C-O phenol-coupling enzyme identified was the cytochrome P450-dependent oxidase berbamunine synthase (CYP80A1) of bisbenzylisoquinoline alkaloid biosynthesis in Berberis cell cultures (5, 6) (Fig. 1). This enzyme is regiospecific, but will accept either (R)- and (S)-N-methylcoclaurine to form R-R and R-S phenol-coupled products. Absolute regio- and stereospecificity is demonstrated in the formation of the lignan (+)-pinoresinol from two molecules of coniferyl alcohol, a reaction guided by dirigent proteins that can be catalyzed by a range of oxidases or oxidants (7). The aporphine alkaloid intramolecular C-C phenol-couple is catalyzed in Coptis japonica cell cultures by the cytochrome P450-dependent oxidase CYP80G2; this enzyme accepts six tetrahydrobenzylisoquinoline alkaloids as substrate (8).Open in a separate windowFIGURE 1.Selected phenol-coupling reactions of alkaloid biosynthesis. Berbamunine synthase (CYP80A1) catalyzes the C-O intermolecular phenol-coupling reaction of bisbenzyisoquinoline alkaloid biosynthesis. (S)-Corytuberine synthase (CYP80G2) catalyzes formation of the intramolecular C-C phenol-couple in magnoflorine biosynthesis. Salutaridine synthase forms the C-C intramolecular phenol-couple of salutaridine in morphine biosynthesis.Morphine has often been described as the king of alkaloids. Although formal syntheses of this powerful analgesic have been reported, yields are low (Ref. 9 and references therein); attempts in organic chemistry to mimic the biosynthetic formation of the C-C phenol-couple of salutaridine (Fig. 1) have been either unsuccessful, yielding rather isoboldine or pallidine (10), or have resulted in very low yield of salutaridine (11) or in a mixture of isoboldine and salutaridine, with the reaction favoring formation of isoboldine by a factor of ∼5 (12). Along with the five stereocenters present in this molecule, the C-C phenol-couple renders a chemical synthesis of morphine commercially unfeasible. The enzyme catalyzing this reaction in planta was sought unsuccessfully for many years and was discovered finally in the opium poppy Papaver somniferum to be a cytochrome P450-dependent oxidase that stereo- and regiospecifically produces salutaridine by C-C phenol-coupling of (R)-reticuline (Fig. 1) (1, 13). The native enzyme salutaridine synthase was unstable, which precluded protein purification for further characterization.Herein, we describe the identification and functional expression of opium poppy salutaridine synthase, a member of the cytochrome P450 family, in Spodoptera frugiperda Sf9 cells. The recombinant enzyme was sufficiently stable in insect cell culture to be characterized with respect to substrate specificity and steady state kinetic values. Recombinant salutaridine synthase converted (R)-reticuline exclusively to salutaridine and (R)-norreticuline exclusively to norsalutaridine (N-demethylsalutaridine).     

The biosynthesis of papaverine proceeds via (S)-reticuline

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 3′ position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine.     

Stereoselective Formation and Metabolism of 4-Hydroxy-Retinoic Acid Enantiomers by Cytochrome P450 Enzymes

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.     

Benzylisoquinoline alkaloid biosynthesis in opium poppy

Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.     

Quantitative <Superscript>1</Superscript>H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

Background  

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (¹H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor.     

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2′-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF_nnic of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.     

Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding

Morphine biosynthesis was genetically engineered in an industrial elite line of the opium poppy (Papaver somniferum L.), to modify the production of alkaloids in plants. The cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B3) lies on the pathway to the benzylisoquinoline alkaloid branch point intermediate (S)-reticuline. Overexpression of cyp80b3 cDNA resulted in an up to 450% increase in the amount of total alkaloid in latex. This increase occurred either without changing the ratio of the individual alkaloids, or together with an overall increase in the ratio of morphine. Correspondingly, antisense-cyp80b3 cDNA expressed in opium poppy caused a reduction of total alkaloid in latex up to 84%, suggesting that the observed phenotypes were dependent on the presence of the transgene. This study found compelling evidence, that cyp80b3 is a key regulation step in morphine biosynthesis and provides practical means to genetically engineer valuable secondary metabolites in this important medicinal plant.     

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.     

Radioimmunoassay determination of six opium alkaloids and its application to plant screening

Radioimmunoassay procedures were developed for the independent and specific determination of sub-nmole quantities of (S)- and (R)-reticuline, salutaridine, thebaine, codeine and morphine. Assay parameters for all six poppy alkaloids are given and the synthesis of haptens and tracers in the Ci/mmol range is described. These assays were used to screen herbarium material of 100 Papaver species and to analyse P. somniferum plant populations for alkaloid breeding purposes. The time course of alkaloid appearance during the germination of poppy seeds was also studied.