Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617169
Title: Investigating protein-protein interactions in the chlorophyll biosynthesis pathway
Author: Hollingshead, Sarah Louise
ISNI:       0000 0004 5348 8310
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Abstract:
The production of chemical energy from light energy is arguably the most important reaction known, which makes photosynthesis one of the most important processes on Earth. Nearly all life depends on the energy derived from light, and it is this process that supports Earths oxygenated atmosphere. Photosynthesis is initiated by the absorption of light, which is performed by specialised pigment molecules known as the chlorophylls. These are highly specialised pigment molecules that belong to the tetrapyrrole family, which also includes vitamin B12, sirohaem and haem. Chlorophyll is synthesised via a series of chemical reactions collectively known as chlorophyll biosynthesis. Whilst the majority of enzymes required for chlorophyll biosynthesis are known and have, to some extent, been characterised, one enzyme, the magnesium protoporphyrin IX monomethylester cyclase (cyclase), remains an enigma. This enzyme catalyses the formation of the 5th isocyclic ring, altering the colour of the tetrapyrrole molecule from red to green. Despite being known for over 60 years, only one subunit of the cyclase has been identified. This is the catalytic or AcsF subunit, which contains a distinctive di-iron motif and is absolutely conserved across all known oxygenic photosynthetic organisms. One of the focuses of this work was to identify other subunits of the cyclase. This led to the discovery of Ycf54, a protein that is highly conserved among oxygenic photosynthetic organisms, which is essential for chlorophyll accumulation in Synechocystis sp. PCC6803. The latter half of this thesis focuses on investigating the structural and functional characteristics of Synechocystis Ycf54; a protein that had previously not been investigated. This work lead the identification of three residues in Ycf54 (D39, F40 and R82), which are required for protochlorophyllide synthesis and chlorophyll formation in Synechocystis, and the observation that these residues are all required for Ycf54 to interact with the catalytic (AcsF) subunit of the cyclase. Additionally, the crystal structure of wild type Synechocystis Ycf54 to a resolution of 1.2 Å, and the structures of two Ycf54 mutants (A9G and R82A), which have an in vivo phenotype were obtained using molecular replacement. Furthermore this work presents the first investigations into the previously unknown protein Slr0483 in Synechocystis. This protein contains the conserved C-terminal membrane anchoring CAAD (Cyanobacterial Aminoacyl-tRNA synthetases Appended Domain) domain and is found in all of the oxygenic photosynthetic organisms investigated. The experiments reported in this thesis show that Slr0483 is essential for photosystem stability and accumulation in Synechocystis and that this protein interacts with the chlorophyll biosynthesis enzymes protoporphyrin IX oxidase, protoporphyrin IX methyltransferase, the cyclase subunit Sll1214 and the geranylgeranyl reductase ChlP, as well as the haem biosynthesis enzyme ferrochelatase. Leading to the hypothesis that Slr0483 may serve as a membrane anchor, localising the enzymes required for chlorophyll and haem biosynthesis, to the sites where these tetrapyrroles are required in the thylakoid membrane (i.e. next to the sites of photosystem and cytochrome assembly).
Supervisor: Hunter, C. Neil Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.617169  DOI: Not available
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