Engineering of CYP82Y1, a cytochrome P450 monooxygenase: a key enzyme in noscapine biosynthesis in opium poppy.

Protein engineering provides a powerful base for the circumvention of challenges tied with characteristics accountable for enzyme functions. CYP82Y1 introduces a hydroxyl group (-OH) into C1 of N-methylcanadine as the substrate to yield 1-hydroxy-N-methylcanadine. This chemical process has been found to be the gateway to noscapine biosynthesis. Owning to the importance of CYP82Y1 in this biosynthetic pathway, it has been selected as a target for enzyme engineering. The insertion of tags to the N- and C-terminal of CYP82Y1 was assessed for their efficiencies for improvement of the physiological performances of CYP82Y1. Although these attempts achieved some positive results, further strategies are required to dramatically enhance the CYP82Y1 activity. Here methods that have been adopted to achieve a functionally improved CYP82Y1 will be reviewed. In addition, the possibility of recruitment of other techniques having not yet been implemented in CYP82Y1 engineering, including the substitution of the residues located in the substrate recognition site, formation of the synthetic fusion proteins, and construction of the artificial lipid-based scaffold will be discussed. Given the fact that the pace of noscapine synthesis is constrained by the CYP82Y1-catalyzing step, the methods proposed here are capable of accelerating the rate of reaction performed by CYP82Y1 through improving its properties, resulting in the enhancement of noscapine accumulation.

Evolutionary analysis of conserved non-coding elements subsequent to whole-genome duplication in opium poppy.

Whole-genome duplication (WGD) leads to the duplication of both coding and non-coding sequences within an organism's genome, providing an abundant supply of genetic material that can drive evolution, ultimately contributing to plant adaptation and speciation. Although non-coding sequences contain numerous regulatory elements, they have been understudied compared to coding sequences. In order to address this gap, we explored the evolutionary patterns of regulatory sequences, coding sequences and transcriptomes using conserved non-coding elements (CNEs) as regulatory element proxies following the recent WGD event in opium poppy (Papaver somniferum). Our results showed similar evolutionary patterns in subgenomes of regulatory and coding sequences. Specifically, the biased or unbiased retention of coding sequences reflected the same pattern as retention levels in regulatory sequences. Further, the divergence of gene expression patterns mediated by regulatory element variations occurred at a more rapid pace than that of gene coding sequences. However, gene losses were purportedly dependent on relaxed selection pressure in coding sequences. Specifically, the rapid evolution of tissue-specific benzylisoquinoline alkaloid production in P. somniferum was associated with regulatory element changes. The origin of a novel stem-specific ACR, which utilized ancestral cis-elements as templates, is likely to be linked to the evolutionary trajectory behind the transition of the PSMT1-CYP719A21 cluster from high levels of expression solely in P. rhoeas root tissue to its elevated expression in P. somniferum stem tissue. Our findings demonstrate that rapid regulatory element evolution can contribute to the emergence of new phenotypes and provide valuable insights into the high evolvability of regulatory elements.

Rational Engineering of (S)-Norcoclaurine Synthase for Efficient Benzylisoquinoline Alkaloids Biosynthesis.

(S)-Norcoclaurine is synthesized in vivo through a metabolic pathway that ends with (S)-norcoclaurine synthase (NCS). The former constitutes the scaffold for the biosynthesis of all benzylisoquinoline alkaloids (BIAs), including many drugs such as the opiates morphine and codeine and the semi-synthetic opioids oxycodone, hydrocodone, and hydromorphone. Unfortunately, the only source of complex BIAs is the opium poppy, leaving the drug supply dependent on poppy crops. Therefore, the bioproduction of (S)-norcoclaurine in heterologous hosts, such as bacteria or yeast, is an intense area of research nowadays. The efficiency of (S)-norcoclaurine biosynthesis is strongly dependent on the catalytic efficiency of NCS. Therefore, we identified vital NCS rate-enhancing mutations through the rational transition-state macrodipole stabilization method at the Quantum Mechanics/Molecular Mechanics (QM/MM) level. The results are a step forward for obtaining NCS variants able to biosynthesize (S)-norcoclaurine on a large scale.

Contrasting self-recognition rejection systems for self-incompatibility in Brassica and Papaver.

Self-incompatibility (SI) plays a pivotal role in whether self-pollen is accepted or rejected. Most SI systems employ two tightly linked loci encoding highly polymorphic pollen (male) and pistil (female) S-determinants that control whether self-pollination is successful or not. In recent years our knowledge of the signalling networks and cellular mechanisms involved has improved considerably, providing an important contribution to our understanding of the diverse mechanisms used by plant cells to recognise each other and elicit responses. Here, we compare and contrast two important SI systems employed in the Brassicaceae and Papaveraceae. Both use 'self-recognition' systems, but their genetic control and S-determinants are quite different. We describe the current knowledge about the receptors and ligands, and the downstream signals and responses utilized to prevent self-seed set. What emerges is a common theme involving the initiation of destructive pathways that block the key processes that are required for compatible pollen-pistil interactions.

Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence.

Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.

Investigating the Effects of Biogenic Zinc Oxide Nanoparticles Produced Using Papaver somniferum Extract on Oxidative Stress, Cytotoxicity, and the Induction of Apoptosis in the THP-1 Cell Line.

This study investigated the effect of novel zinc oxide nanoparticles (ZnO NPs) biosynthesized employing Papaver somniferum leaf on oxidative stress, necrosis, and apoptosis in the leukemia cancer THP-1 cell. The obtained ZnO was examined using SEM, AFM, and TEM microscopy, which revealed an irregular spherical morphology with a size ranging from 20 to 30 nm, and the UV-vis absorbance revealed a strong absorption peak in the range of 360-370, nm confirming the production of ZnO NPs. THP-1 cells were subjected to an MTT, an EdU proliferation, a lactate dehydrogenase release tests, a reactive oxygen species (ROS) induction experiment, a DAPI staining detection assay, and a flow cytometric analysis for Annexin V to measure the effects of ZnO NPs on cancer cell growth inhibition, apoptosis, and necrosis. Our results show that ZnO NPs inhibit THP-1 line in a concentration-dependent pattern. It was observed that ZnO NPs triggered necrosis (cell death) and apoptosis in the cell line. ZnO NPs massively improved the formation of intracellular ROS, which is crucial in deactivating the development of leukemic cells. In conclusion, ZnO nanoparticles synthesized using Papaver somniferum extract have the ability to inhibit proliferation leukemic cancer cells, making them potential anticancer agents.

Structural Characterization and Molecular Dynamics Study of the REPI Fusion Protein from Papaver somniferum L.

REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.

Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation.

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.

Computational insights into diverse aspects of glutathione S-transferase gene family in Papaver somniferum.

Plant glutathione S-transferases are an ancient protein superfamily having antioxidant activity. These proteins are primarily involved in diverse plant functions such as plant growth and development, secondary metabolism, signaling pathways and defense against biotic and abiotic stresses. The current study aimed to comprehensively identify and characterize the GST gene family in the medicinally important crop Papaver somniferum. A total of 93 GST proteins were identified belonging to eight GST classes and found to be majorly localized in the cytoplasm. All GST genes were found on eleven opium chromosomes. Gene duplication analysis showed segmental duplication as a key factor for opium GST gene family expansion under strong purifying selection. Phylogenetic analysis with gymnosperm, angiosperm and bryophyte revealed the evolution of GSTs earlier than their division into separate groups and also prior to the divergence of monocot and dicot. The secondary structure prediction showed the dominance of α-helices indicative of PsomGSTs as structurally stable and elastic proteins. Gene architecture showed the conservation of number of exons across the classes. MEME analysis revealed only a few class specific and many across class conserved motifs. Ser was found to be the active site residue of tau, phi, theta and zeta class and Cys was catalytic residue of DHAR, lambda and GHR class. Promoter analyses identified many cis-acting regulatory elements related to hormonal, cellular, stress and light response functions. Ser was the key phosphorylation site. Only three glycosylation sites were found across the 93 PsomGSTs. 3D structure prediction was also performed and was validated. Interactome analyses revealed the correlation of PsomGSTs with glutathione metabolizing proteins. Gene enrichment analysis and KEGG pathway analyzed the involvement of PsomGSTs in three major pathways i.e. glutathione metabolism, tyrosine metabolism and ascorbate metabolism. The outcome revealed high model quality of PsomGSTs. The results of the current study will be of potential significance to understand the functional and structural importance of the GST gene family in opium, a medicinally important crop.

Depletion plays a pivotal role in self-incompatibility, revealing a link between cellular energy status, cytosolic acidification and actin remodelling in pollen tubes.

Self-incompatibility (SI) involves specific interactions during pollination to reject incompatible ('self') pollen, preventing inbreeding in angiosperms. A key event observed in pollen undergoing the Papaver rhoeas SI response is the formation of punctate F-actin foci. Pollen tube growth is heavily energy-dependent, yet ATP levels in pollen tubes have not been directly measured during SI. Here we used transgenic Arabidopsis lines expressing the Papaver pollen S-determinant to investigate a possible link between ATP levels, cytosolic pH ([pH]cyt ) and alterations to the actin cytoskeleton. We identify for the first time that SI triggers a rapid and significant ATP depletion in pollen tubes. Artificial depletion of ATP triggered cytosolic acidification and formation of actin aggregates. We also identify in vivo, evidence for a threshold [pH]cyt of 5.8 for actin foci formation. Imaging revealed that SI stimulates acidic cytosolic patches adjacent to the plasma membrane. In conclusion, this study provides evidence that ATP depletion plays a pivotal role in SI upstream of programmed cell death and reveals a link between the cellular energy status, cytosolic acidification and alterations to the actin cytoskeleton in regulating Papaver SI in pollen tubes.

A functionally conserved STORR gene fusion in Papaver species that diverged 16.8 million years ago.

The STORR gene fusion event is considered essential for the evolution of the promorphinan/morphinan subclass of benzylisoquinoline alkaloids (BIAs) in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline essential for their biosynthesis. Here, we show that of the 12 Papaver species analysed those containing the STORR gene fusion also contain promorphinans/morphinans with one important exception. P. californicum encodes a functionally conserved STORR but does not produce promorphinans/morphinans. We also show that the gene fusion event occurred only once, between 16.8-24.1 million years ago before the separation of P. californicum from other Clade 2 Papaver species. The most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs, raising the possibility that STORR, once evolved, contributes to the biosynthesis of more than just the promorphinan/morphinan subclass of BIAs in the Papaveraceae.

Self-incompatibility requires GPI anchor remodeling by the poppy PGAP1 ortholog HLD1.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are tethered to the outer leaflet of the plasma membrane where they function as key regulators of a plethora of biological processes in eukaryotes. Self-incompatibility (SI) plays a pivotal role regulating fertilization in higher plants through recognition and rejection of "self" pollen. Here, we used Arabidopsis thaliana lines that were engineered to be self-incompatible by expression of Papaver rhoeas SI determinants for an SI suppressor screen. We identify HLD1/AtPGAP1, an ortholog of the human GPI-inositol deacylase PGAP1, as a critical component required for the SI response. Besides a delay in flowering time, no developmental defects were observed in HLD1/AtPGAP1 knockout plants, but SI was completely abolished. We demonstrate that HLD1/AtPGAP1 functions as a GPI-inositol deacylase and that this GPI-remodeling activity is essential for SI. Using GFP-SKU5 as a representative GPI-AP, we show that the HLD1/AtPGAP1 mutation does not affect GPI-AP production and targeting but affects their cleavage and release from membranes in vivo. Our data not only implicate GPI-APs in SI, providing new directions to investigate SI mechanisms, but also identify a key functional role for GPI-AP remodeling by inositol deacylation in planta.

Phloem-specific localization of benzylisoquinoline alkaloid metabolism in opium poppy.

Opium poppy is the only commercial source of the narcotic analgesics morphine and codeine, and semi-synthetic derivatives of the natural opiate precursor thebaine, including oxycodone and the opioid antagonist naloxone. The plant also accumulates the vasodilator and antitussive agents papaverine and noscapine, respectively, which together with morphine, codeine and thebaine comprise the major benzylisoquinoline alkaloids (BIAs) in opium poppy. A majority of enzymes involved in the highly branched BIA metabolism in opium poppy have now been discovered, with many specifically localized to sieve elements of the phloem based on immunofluorescence labeling techniques. Transcripts corresponding to sieve element-localized biosynthetic enzymes were detected in companion cells, as expected. The more recent application of shotgun proteomics has shown that several enzymes operating late in the morphine and noscapine biosynthetic pathways occur primarily in laticifers that are adjacent or proximal to sieve elements. BIA biosynthesis and accumulation in opium poppy involves three phloem cell types and implicates the translocation of key pathway intermediates between sieve elements and laticifers. The recent isolation of uptake transporters associated with laticifers supports an apoplastic rather than a symplastic route for translocation. In spite of the extensive elucidation of BIA biosynthetic enzymes in opium poppy, additional transporters and other auxiliary proteins are clearly necessary to support the complex spatial organization and dynamics involved in product formation and sequestration. In this review, we provide an update of BIA metabolism in opium poppy with a focus on the role of phloem in the biosynthesis of the major alkaloids.

Insights into opium poppy (Papaver spp.) genetic diversity from genotyping-by-sequencing analysis.

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and a versatile model system to study secondary metabolism. However, our knowledge of its genetic diversity is limited, restricting utilization of the available germplasm for research and crop improvement. We used genotyping-by-sequencing to investigate the extent of genetic diversity and population structure in a collection of poppy germplasm consisting of 91 accessions originating in 30 countries of Europe, North Africa, America, and Asia. We identified five genetically distinct subpopulations using discriminate analysis of principal components and STRUCTURE analysis. Most accessions obtained from the same country were grouped together within subpopulations, likely a consequence of the restriction on movement of poppy germplasm. Alkaloid profiles of accessions were highly diverse, with morphine being dominant. Phylogenetic analysis identified genetic groups that were largely consistent with the subpopulations detected and that could be differentiated broadly based on traits such as number of branches and seed weight. These accessions and the associated genotypic data are valuable resources for further genetic diversity analysis, which could include definition of poppy core sets to facilitate genebank management and use of the diversity for genetic improvement of this valuable crop.

Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.

Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.

Three chromosome-scale Papaver genomes reveal punctuated patchwork evolution of the morphinan and noscapine biosynthesis pathway.

For millions of years, plants evolve plenty of structurally diverse secondary metabolites (SM) to support their sessile lifestyles through continuous biochemical pathway innovation. While new genes commonly drive the evolution of plant SM pathway, how a full biosynthetic pathway evolves remains poorly understood. The evolution of pathway involves recruiting new genes along the reaction cascade forwardly, backwardly, or in a patchwork manner. With three chromosome-scale Papaver genome assemblies, we here reveal whole-genome duplications (WGDs) apparently accelerate chromosomal rearrangements with a nonrandom distribution towards SM optimization. A burst of structural variants involving fusions, translocations and duplications within 7.7 million years have assembled nine genes into the benzylisoquinoline alkaloids gene cluster, following a punctuated patchwork model. Biosynthetic gene copies and their total expression matter to morphinan production. Our results demonstrate how new genes have been recruited from a WGD-induced repertoire of unregulated enzymes with promiscuous reactivities to innovate efficient metabolic pathways with spatiotemporal constraint.

Identification of novel anti-cancer agents by the synthesis and cellular screening of a noscapine-based library.

Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine.

Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl.

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. The effects of induced polyploidy as well as the effect of methyl Jasmonate (MeJA) elicitation on the root production of thebaine and on the expression of five alkaloid biosynthesis related genes were studied. The in vitro tetraploidy induction caused a significant increased expression of norcoclaurine synthase (NCS) and salutaridinol (SAT), and a significant decreased expression of berberine bridge enzyme (BBE) in the leaves. In the root tissues, the BBE, NCS, and SAT showed an increased expression in tetraploid plants, while codeinone reductase (COR) showed a decreased expression. A similar alteration pattern was found in mixoploid plants when compared to their diploid counterparts. MeJA at concentrations of 0.1 and 0.5 mM caused a remarkable increase in the thebaine content in the roots of treated plants, where the highest thebaine content was identified in plants elicited with 0.5 mM MeJA. Elicitation treatment caused a substantial increase in the expression of NCS and SAT in the leaves, while it had no major effect on BBE, codeine 3-O-demethylase (CODM) and COR. Expression analysis in the roots showed that MeJA caused a significant increase in the expression of only BBE and NCS, while expression of other studied genes remained unchanged. Our results may be exploited for improved thebaine production and the processing of Persian poppy.

Structural insights into thebaine synthase 2 catalysis.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.

Molecular Network-Guided Alkaloid Profiling of Aerial Parts of Papaver nudicaule L. Using LC-HRMS.

Papaver nudicaule L. (Iceland poppy) is widely used for ornamental purposes. A previous study demonstrated the alleviation of lipopolysaccharide-induced inflammation mediated by P. nudicaule extract through nuclear factor-kappa B and signal transducer and activator of transcription 3 inactivation. As isoquinoline alkaloids are chemical markers and bioactive constituents of Papaver species, the present study investigated the alkaloid profile of aerial parts of five P. nudicaule cultivars with different flower colors and a P. rhoeas cropped for two years. A combination of liquid chromatography high-resolution mass spectrometry and molecular networking was used to cluster isoquinoline alkaloids in the species and highlight the possible metabolites. Aside from the 12 compounds, including rotundine, muramine, and allocryptopine, identified from Global Natural Products Social library and reported information, 46 structurally related metabolites were quantitatively investigated. Forty-two and 16 compounds were proposed for chemical profiles of P. nudicaule and P. rhoeas, respectively. Some species-specific metabolites showed similar fragmentation patterns. The alkaloid abundance of P. nudicaule differed depending on the flower color, and the possible chemical markers were proposed. These results show that molecular networking-guided dereplication allows investigation of unidentified metabolites. The derived chemical profile may facilitate evaluation of P. nudicaule quality for pharmacological applications.