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Biological evaluation of the carotenoid cleavage dioxygenase family and apocarotenoid signalling via chemical genetics

Reference	BB/D005787/1
Principal Investigator / Supervisor	Professor Andrew Thompson
Co-Investigators / Co-Supervisors	Professor Timothy Bugg, Dr Martin Sergeant
Institution	University of Warwick
Department	Warwick HRI
Funding type	Research
Value (£)	434,921
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	31/05/2009
Duration	42 months

Abstract

The diversity and importance of carotenoid cleavage products (apocarotenoids) as signals in numerous biological systems has become increasingly apparent in recent years. Equally, the size and diversity of the recently discovered family of carotenoid cleavage dioxygenases (CCDs) is rapidly increasing and enzymes have now been described that cleave carotenoids at five different positions in the polyene chain. This proposal concerns the application of chemical genetics to study this novel family of enzymes, and their involvement in the biosynthesis of plant, animal and bacterial signalling molecules. The proposal brings together a biological chemist with a background in mechanistic enzymology of dioxygenases, and a plant scientist with a research interest in biosynthesis of the apocarotenoid plant hormone, abscisic acid. In many cases the precise function of specific CCDs is unknown, and in some cases the identity of the signalling molecule has not yet been established. Using the chemical genetics approach we aim (i) to determine the biological function of specific CCDs and their apocarotenoid products in a diverse range of biological systems, and (ii) to determine the in vivo substrates and products of specific CCDs. We will synthesize chemical libraries aimed at selectively inhibiting the activity of particular classes of CCD activity. Synthesis will be directed to produce inhibitors specific to different carotenoid cleavage positions using as a starting point a known inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED). This inhibitor, abamine, is a putative mimic of a carbocation intermediate during cleavage at the C11,C12 position and is specific to the 9-cis isomer. Alternative structures will be aimed at carbocation intermediates for other cleavage positions and all-trans isomers. An alternative approach of incorporating a metal chelating group into substrate analogs will also be pursued. Chemical libraries will then be screened against a panel of diverseCCDs, chosen to represent a wide range of carotenoid cleavage positions and taxonomic kingdoms. Screening will utilize E.coli strains that express enzymes for CCD substrate synthesis, accumulate beta-carotene, lycopene or zeaxanthin and exhibit characteristic colours. Expression of CCDs in these strains reduces colour, and application of active inhibitors will restore colour to provide a facile visual screen. It is not currently possible to produce E.coli strains that accumulate 9-cis-epoxycarotenoids so the screen for NCED inhibitors will be based on promotion of germination in seed where germination is inhibited by excessive abscisic acid produced from an NCED transgene. In vitro enzyme assays with recombinant protein would provide alternative chemical library screens and will be used for kinetic and mechanistic studies of inhibitor action and selectivity. Once selective inhibitors are identified they will be used to evaluate biological function of CCDs and apocarotenoids. A diverse range of biological systems will be challenged with specific inhibitors and then assessed for physiological and/or developmental effects. Alterations to carotenoid and apocarotenoid profiles will be assessed using HPLC, GC-MS, electospray-MS and LC-SPE-NMR. The biological systems investigated, in some cases with the help of collaborators within The University of Warwick, will be: (i) control of growth, dormancy and stomatal conductance by abscisic acid, (ii) the involvement of apocarotenoids in arbuscular mycorrhizal associations, (iii) control of flower volatiles, and (iv) the unknown function of apocarotenoids in cyanobacteria. We will also provide inhibitors to two collaborating groups with expertise in a novel and poorly characterised apocarotenoid plant hormone controlling branching (York), and in the role of carotenoid cleavage/retinoic acid metabolism in carcinogenesis (Newcastle).

Summary

Aims This project aims to use a new scientific approach called 'chemical genetics' to study the function of a specific type of enzyme known as carotenoid cleavage dioxygenases (CCDs). We will make synthetic chemicals designed to only block the action of CCDs, apply them to range of living organisms, and then see how they respond. This will tell us about the role CCDs have, and about the chemical reactions they influence. Context Carotenoids are pigments made in plants, fungi and bacteria, and include the chemicals that make carrots orange and tomatoes red. Living creatures evolved to make carotenoids probably because they are useful for protection against both sunlight and reactive chemicals such as free-radicals. A diverse range of organisms have found further uses for carotenoids by cutting them up into smaller pieces that often act as signalling molecules and facilitate communication between cells, organs, individuals or species. These are called apocarotenoids (apo means 'detached from'). For example, humans need carotenoids (e.g. provitamin A) in the diet because they are broken down into apocarotenoids that are essential for vision and also correct development of the brain and limbs. Plants use apocarotenoids to help them respond to stress, to control their branching, for germination and to signal to pollinating insects. Apocarotenoids are also found in bacteria, insects and fungi but in many cases we do not know what their functions are and it is likely that there are more apocarotenoid signalling pathways still to be found. It has recently been discovered that the cutting-up of carotenoids is controlled by a large family of enzymes known as the carotenoid cleavage dioxygenases or CCDs. Carotenoid molecules generally contain a long chain of 40 carbon atoms, and vary in their structures at the end of the chain, and it is known that different types of CCD can cut the carbon chain in different places to produce apocarotenoids of different sizes and structures. Applications and Benefits Several research groups have discovered different CCDs by studying mutant plants that lack one CCD, or by making enzymes in bacteria and then testing their activity. However, we do not fully understand the function of many of these enzymes or the apocarotenoids they produce in the context of complex living organisms. During this project we will make and use chemical tools to investigate known examples of CCDs and to look for new apocarotenoid functions. Because apocarotenoids are important in such a wide range of organisms and processes there will be many different applications and benefits. One example is in the treatment of cancer: it is suggested that in some cases (e.g. in people who smoke) the action of CCDs can produce apocarotenoids that increase the risk of cancer. Controlling the balance of activity between different CCDs may provide a treatment to reduce risk of cancer. Also in vertebrates the ability to block CCDs at particular stages of development using chemicals will help us to understand the role of apocarotenoids in controlling brain and limb formation. In plants, the ability to block particular CCDs may provide a means to understand and control (i) growth during drought stress, (ii) germination of seeds, (iii) the way that stems branch, (iv) symbiotic interactions between fungi and plants and (v) the aroma of flowers and fruit. In bacteria the functions of apocarotenoids are almost entirely unknown and chemical inhibitors will provide a powerful way to understand function.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Industrial Biotechnology, Microbiology, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	Selective Chemical Intervention In Biological Systems (SCIBS) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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