Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.699602
Title: Duplication of alcohol dehydrogenases unlocks the chemical diversity of the medicinal plant Catharanthus roseus
Author: Stavrinides, Anna
ISNI:       0000 0004 5990 3992
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Thesis embargoed until 28 Nov 2020
Access from Institution:
Abstract:
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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.699602  DOI: Not available
Share: