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-β-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.
Allopolyploidization is a widespread phenomenon known to generate novel phenotypes by merging evolutionarily distinct parental genomes and regulatory networks in a single nucleus. The objective of ...this study was to investigate the transcriptional regulation associated with phenotypic novelty in coffee beans of the allotetraploid Coffea arabica.
A genome-wide comparative transcriptomic analysis was performed in C. arabica and its two diploid progenitors, C. canephora and C. eugenioides. Gene expression patterns and homeologue expression were studied on seeds at five different maturation stages. The involvement of homeologue expression bias (HEB) in specific traits was addressed both by functional enrichment analyses and by the study of gene expression in the caffeine and chlorogenic acid biosynthesis pathways.
Expression-level dominance in C. arabica seed was observed for most of the genes differentially expressed between the species. Approximately a third of the genes analysed showed HEB. This proportion increased during seed maturation but the biases remained equally distributed between the sub-genomes. The relative expression levels of homeologues remained relatively constant during maturation and were correlated with those estimated in leaves of C. arabica and interspecific hybrids between C. canephora and C. eugenioides. Functional enrichment analyses performed on genes exhibiting HEB enabled the identification of processes potentially associated with physiological traits. The expression profiles of the genes involved in caffeine biosynthesis mirror the differences observed in the caffeine content of mature seeds of C. arabica and its parental species.
Neither of the two sub-genomes is globally preferentially expressed in C. arabica seeds, and homeologues appear to be co-regulated by shared trans-regulatory mechanisms. The observed HEBs are thought to be a legacy of gene expression differences inherited from diploid progenitor species. Pre-existing functional divergences between parental species appear to play an important role in controlling the phenotype of C. arabica seeds.
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.
Abstract
In contrast to desiccation-tolerant ‘orthodox’ seeds, so-called ‘intermediate’ seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate ...seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses.
Seeds of coffee species that are more tolerant to desiccation display a specific late maturation transcriptional program that primes them to better cease respiration and avoid oxidative stress upon dehydration.
Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, is a serious, recently emerged disease of barley in Europe and other temperate regions. This study investigated the trade off ...between strong resistance to powdery mildew conferred by mlo mutant alleles and increased susceptibility to RLS. In field trials and seedling tests, the presence of mlo alleles increased severity of RLS. Genetic analysis of a doubled-haploid population identified one quantitative trait locus for susceptibility to RLS, colocalizing with the mlo-11 allele for mildew resistance. The effect of mlo-11 on RLS severity was environmentally sensitive. Analysis of near-isogenic lines of different mlo mutations in various genetic backgrounds confirmed that mlo alleles increased RLS severity in seedlings and adult plants. For mlo resistance to mildew to be fully effective, the genes ROR1 and ROR2 are required. RLS symptoms were significantly reduced on mlo-5 ror double mutants but fungal DNA levels remained as high as in mlo-5 single mutants, implying that ror alleles modify the transition of the fungus from endophytism to necrotrophy. These results indicate that the widespread use of mlo resistance to control mildew may have inadvertently stimulated the emergence of RLS as a major disease of barley.
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.
Cryptic and unnatural natural products: A short‐chain alcohol dehydrogenase from plant producers of monoterpene indole alkaloids (C. roseus and R. serpentina) reduces strictosidine aglycone to form an alkaloid that does not correspond to any previously reported compound.
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.
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•Tetrahydroalstonine synthase catalyzes the formation of a plant-derived alkaloid•Tetrahydroalstonine synthase is localized to the nucleus•Tetrahydroalstonine synthase and the preceding pathway enzyme interact•Discovery of a gene controlling structural diversity of monoterpene indole alkaloids
How plants transform the central biosynthetic intermediate strictosidine into thousands of divergent alkaloids has remained unresolved. Stavrinides et al. discover a nuclear-localized alcohol dehydrogenase homolog responsible for conversion of strictosidine aglycone to tetrahydroalstonine that appears to interact with an upstream pathway enzyme.
This thesis details the discovery and characterisation of biosynthetic enzymes implicated in the monoterpene indole alkaloid (MIA) pathway of the medicinal plant Catharanthus roseus. The MIA pathway ...is characterised by a plethora of different carbon skeletons, which are derived from the central pathway intermediate strictosidine aglycon. Given the biological importance of these compounds, there is great interest in identifying the enzymes that catalyse the formation of these different carbon skeletons, as well as understanding the mechanistic basis for how the pathway is controlled at this critical step in the pathway. The discovery of the first enzyme capable of reducing the reactive aglycon intermediate to a heteroyohimbine-type MIA (tetrahydroalstonine) opened the door to discovery of many other heteroyohimbine synthases (HYS, Chapter 2). These enzymes share a degree of sequence identity and are all members of the Medium-Chain Dehydrogenase/Reductases. Interestingly, these HYSs catalysed the formation of different ratios of several heteroyohimbine stereoisomers. A detailed mutation screen, together with protein crystallography, deuterium labelling, and in silico docking enabled us to propose a catalytic mechanism for these enzymes and how the ratio of products is controlled (Chapter 3). An investigation into a different family of reductases, the Short-Chain Dehydrogenase/Reductases, revealed another enzyme capable of reducing the reactive strictosidine aglycon intermediate (Chapter 4). After extensive NMR characterisation, the enzymatic product was found to possess an unusual carbon skeleton different to that of the heteroyohimbines. Characterisation of the phylogeny of these enzymes revealed that they have undergone numerous duplication events (Chapter 5). The HYSs appear to have undergone multiple duplications and neofunctionalisation that has given rise to at least one other biosynthetic enzyme which acts in one of the downstream MIA pathway branches. A study of a large reductase duplication locus provides evidence that pathway clustering in plants arises through translocation of biosynthetic genes. The discovery of these reductases has provided us with an unprecedented opportunity to study the dynamics of the branch-point of the MIA pathway. These discoveries constitute an important step towards the elucidation of the MIA pathway in C. roseus and in the many related MIA producing plants.
This thesis details the discovery and characterisation of biosynthetic enzymes implicated in the monoterpene indole alkaloid (MIA) pathway of the medicinal plant Catharanthus roseus. The MIA pathway ...is characterised by a plethora of different carbon skeletons, which are derived from the central pathway intermediate strictosidine aglycon. Given the biological importance of these compounds, there is great interest in identifying the enzymes that catalyse the formation of these different carbon skeletons, as well as understanding the mechanistic basis for how the pathway is controlled at this critical step in the pathway. The discovery of the first enzyme capable of reducing the reactive aglycon intermediate to a heteroyohimbine-type MIA (tetrahydroalstonine) opened the door to discovery of many other heteroyohimbine synthases (HYS, Chapter 2). These enzymes share a degree of sequence identity and are all members of the Medium-Chain Dehydrogenase/Reductases. Interestingly, these HYSs catalysed the formation of different ratios of several heteroyohimbine stereoisomers. A detailed mutation screen, together with protein crystallography, deuterium labelling, and in silico docking enabled us to propose a catalytic mechanism for these enzymes and how the ratio of products is controlled (Chapter 3). An investigation into a different family of reductases, the Short-Chain Dehydrogenase/Reductases, revealed another enzyme capable of reducing the reactive strictosidine aglycon intermediate (Chapter 4). After extensive NMR characterisation, the enzymatic product was found to possess an unusual carbon skeleton different to that of the heteroyohimbines. Characterisation of the phylogeny of these enzymes revealed that they have undergone numerous duplication events (Chapter 5). The HYSs appear to have undergone multiple duplications and neofunctionalisation that has given rise to at least one other biosynthetic enzyme which acts in one of the downstream MIA pathway branches. A study of a large reductase duplication locus provides evidence that pathway clustering in plants arises through translocation of biosynthetic genes. The discovery of these reductases has provided us with an unprecedented opportunity to study the dynamics of the branch-point of the MIA pathway. These discoveries constitute an important step towards the elucidation of the MIA pathway in C. roseus and in the many related MIA producing plants.
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