The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae.

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.

Deciphering and reprogramming the cyclization regioselectivity in bifurcation of indole alkaloid biosynthesis.

The metabolism of monoterpene indole alkaloids (MIAs) is an outstanding example of how plants shape chemical diversity from a single precursor. Here we report the discovery of novel enzymes from the Alstonia scholaris tree, a cytochrome P450, an NADPH dependent oxidoreductase and a BAHD acyltransferase that together synthesize the indole alkaloid akuammiline with a unique methanoquinolizidine cage structure. The two paralogous cytochrome P450 enzymes rhazimal synthase (AsRHS) and geissoschizine oxidase (AsGO) catalyse the cyclization of the common precursor geissoschizine and they direct the MIA metabolism towards to the two structurally distinct and medicinally important MIA classes of akuammilan and strychnos alkaloids, respectively. To understand the pathway divergence, we investigated the catalytic mechanism of the two P450 enzymes by homology modelling and reciprocal mutations. Upon conducting mutant enzyme assays, we identified a single amino acid residue that mediates the space in active sites, switches the enzymatic reaction outcome and impacts the cyclization regioselectivity. Our results represent a significant advance in MIA metabolism, paving the way for discovery of downstream genes in akuammilan alkaloid biosynthesis and facilitating future synthetic biology applications. We anticipate that our work presents, for the first time, insights at the molecular level for plant P450 catalytic activity with a significant key role in the diversification of alkaloid metabolism, and provides the basis for designing new drugs.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

Discovery of a Short-Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid.

Plant monoterpene indole alkaloids, a large class of natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short-chain alcohol dehydrogenase (SDR) from plant producers of monoterpene indole alkaloids (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyse. This discovery further highlights how a sequence-based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.

A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate.

Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism-an alcohol dehydrogenase and a cytochrome P450-produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-ß-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.

Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold.

New developments in engineering plant metabolic pathways.

Plants contain countless metabolic pathways that are responsible for the biosynthesis of complex metabolites. Armed with new tools in sequencing and bioinformatics, the genes that encode these plant biosynthetic pathways have become easier to discover, putting us in an excellent position to fully harness the wealth of compounds and biocatalysts (enzymes) that plants provide. For overproduction and isolation of high-value plant-derived chemicals, plant pathways can be reconstituted in heterologous hosts. Alternatively, plant pathways can be modified in the native producer to confer new properties to the plant, such as better biofuel production or enhanced nutritional value. This perspective highlights a range of examples that demonstrate how the metabolic pathways of plants can be successfully harnessed with a variety of metabolic engineering approaches.

Unprecedented Utilization of Pelargonidin and Indole for the Biosynthesis of Plant Indole Alkaloids.

Nudicaulins are a group of indole alkaloid glycosides responsible for the color of yellow petals of Papaver nudicaule (Iceland poppy). The unique aglycone scaffold of these alkaloids attracted our interest as one of the most unusual flavonoid-indole hybrid structures that occur in nature. Stable isotope labeling experiments with sliced petals identified free indole, but not tryptamine or l-tryptophan, as one of the two key biosynthetic precursors of the nudicaulin aglycone. Pelargonidin was identified as the second key precursor, contributing the polyphenolic unit to the nudicaulin molecule. This finding was inferred from the temporary accumulation of pelargonidin glycosides in the petals during flower bud development and a drop at the point in time when nudicaulin levels start to increase. The precursor-directed incorporation of cyanidin into a new 3'-hydroxynudicaulin strongly supports the hypothesis that anthocyanins are involved in the biosynthesis of nudicaulins.

Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channeling in secondary metabolism.

The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway.

Biosynthesis of Nudicaulins: A (13) CO2 -pulse/chase labeling study with Papaver nudicaule.

Nudicaulins are unique alkaloids responsible for the yellow color of the petals of some papaveraceaous plants. To elucidate the unknown biosynthetic origin of the skeleton, a (13) CO2 -pulse/chase experiment was performed with growing Papaver nudicaule plants. (13) C NMR analysis revealed more than 20 multiple (13) C-enriched isotopologues in nudicaulins from the petals of (13) CO2 -labeled plants. The complex labeling pattern was compared with the isotopologue composition of a kaempferol derivative that was isolated from petals of the same (13) CO2 -labeled plants. The deconvolution of the labeling profiles indicated that the nudicaulin scaffold is assembled from products or intermediates of indole metabolism, the phenylpropanoid pathway, and the polyketide biosynthesis. Naringenin-type compounds and tryptophan/tryptamine are potential substrates for the condensation reaction finally generating the aglycone skeleton of nudicaulins.

Occurrence of nudicaulin structural variants in flowers of papaveraceous species.

The intense color of yellow Papaver nudicaule flowers is conferred by the presence of nudicaulins, a group of alkaloids with a unique pentacyclic skeleton composed of an indole ring and a polyphenolic moiety. Petals from eight different Papaveraceae species composed of different color varieties were probed for the presence of nudicaulins. In addition to their occurrence in yellow P. nudicaule flowers, nudicaulins I-VIII were detected and quantified in orange flowers of P. nudicaule, and in yellow and orange Papaver alpinum flowers. Meconopsis cambrica petals showed a divergent nudicaulin spectrum, with compounds having an attached 3-hydroxy-3-methyl-glutaryl group (HMG) instead of a malonyl unit at one of the glucose units. Flavonols and anthocyanins that accompany nudicaulins were identified. The taxonomical significance of the occurrence of nudicaulins is briefly discussed.

Concentration kinetics of secoisolariciresinol diglucoside and its biosynthetic precursor coniferin in developing flaxseed.

INTRODUCTION: In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of secoisolariciresinol diglucoside (SDG), which is of great interest because of its potential health benefits for human beings. The information about the kinetics of SDG formation during flaxseed development is rare and incomplete. OBJECTIVE: In this study, a reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed to quantify SDG and coniferin, a key biosynthetic precursor of SDG in flaxseed. METHODOLOGY: Seeds from different developmental stages, which were scaled by days after flowering (DAF), were harvested. After alkaline hydrolysis, the validated HPLC method was applied to determine SDG and coniferin concentrations of flaxseed from different developing stages. RESULTS: Coniferin was found in the entire capsule as soon as flowering started and became undetectable 20 DAF. SDG was detected 6 DAF, and the concentration increased until maturity. On the other hand, the SDG amount in a single flaxseed approached the maximum around 25 DAF, before desiccation started. Concentration increase between 25 DAF and 35 DAF can be attributed to corresponding seed weight decrease. CONCLUSION: The biosynthesis of coniferin is not synchronous with that of SDG. Hence, the concentrations of SDG and coniferin change during flaxseed development.

Nudicaulins, yellow flower pigments of Papaver nudicaule: revised constitution and assignment of absolute configuration.

The constitution of the aglycon of nudicaulin has been revised to be a pentacyclic indole alkaloid. The relative and absolute configurations of the two diastereomers, nudicaulins I (3a) and II (3b), have been assigned by NMR, conformational analyses, and interpretation of the experimental ECD spectra by quantum-chemical calculations.

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups.

The (1)H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD(3)OH solutions indicate significantly deshielded signals in the region of 14-15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional (1)H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14-15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV-vis and LC/MS measurements.

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS.

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.