Duplication of alcohol dehydrogenases unlocks the chemical diversity of the medicinal plant Catharanthus roseus

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

Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism

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

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Seed comparative genomics in three coffee species identify desiccation tolerance mechanisms in intermediate seeds

International audienceIn 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 slowdown 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

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

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