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Characterisation of Chlorophyll Synthases from Cyanobacteria and Plants

Proctor, Matthew Stephen (2018) Characterisation of Chlorophyll Synthases from Cyanobacteria and Plants. PhD thesis, University of Sheffield.

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Abstract

During the process of photosynthesis, oxygenic photosynthetic organisms utilise chlorophyll (Chl) molecules, spatially organised within membrane associated protein complexes called photosystems, to capture light from the sun and convert it into chemical energy. Chls are tetrapyrrole molecules featuring a fifth ring, a central Mg2+ ion and a hydrophobic phytyl tail. The integral membrane protein chlorophyll synthase (ChlG) catalyses the addition of the tail to the pyrrole ring. In the model photosynthetic cyanobacteria Synechocystis, ChlG forms a protein-pigment complex with high-light inducible proteins C and D (HliC/HliD), photosystem II assembly factor Ycf39, the YidC/Alb3 insertase and pigments zeaxanthin, myxoxanthophyll, β-carotene and Chl. This complex is postulated to act at the interface between Chl biosynthesis and photosystem assembly, coordinating co-translational insertion of de novo Chl molecules into Chl-binding proteins in a poorly understood process. To gain an insight into the ubiquity of the ChlG complex in higher photosynthetic organisms, ChlG genes from a plant and algae were FLAG-tagged and heterologously expressed in Synechocystis. The eukaryotic ChlG homologues could complement the function of the native bacterial protein but did not associate with HliD or Ycf39, maintaining an association only with YidC. This indicates that the ChlG-YidC/Alb3 association may be evolutionarily conserved in algae and higher plants. Abolishing the synthesis of zeaxanthin and myxoxanthophyll in Synechocystis prevented association of HliD and Ycf39 with ChlG, indicating that these carotenoids mediate formation of the ChlG complex. Selective abolishment of myxoxanthophyll restored binding of these proteins, suggesting that zeaxanthin alone can facilitate the ChlG-HliD-Ycf39 interaction. Structural investigation by chemical cross-linking revealed sites of interaction between members of the ChlG complex. These were found to be confined to the cytoplasm. The N-terminal domain of ChlG was the only region of the enzyme found to interact with its partner proteins. The results enabled the generation of a model of the ChlG complex. The N-terminus of ChlG was sequentially truncated to investigate the importance of this domain to formation of the ChlG complex. Four truncations were made, removing 11, 23, 32 and 39 residues from the N-terminus, up until the start of the first predicted transmembrane helix. While the binding of YidC, HliD and Ycf39 was not impeded in any case, the enzyme activity of ChlG reduced as the truncations became larger. ChlGs lacking 32 or more residues were unable to complement the function of the native enzyme in vivo and showed significantly reduced activity in vitro. The results indicate that the N-terminal domain of ChlG is important for facilitating its catalytic activity. A method for the rapid generation and in vitro testing of point mutations to the function of Arabidopsis ChlG was developed. This involved optimising production of ChlG in E. coli in addition to developing a method for producing the enzymes substrate, chlorophyllide. A ChlG model was generated using the crystal structure of a related protein, UbiA, as a template. The optimised methods were used to generate six point mutations, predicted from the model and sequence alignments of ChlG with UbiA to be important for enzyme activity or substrate binding. Three of the mutants were devoid of activity, demonstrating the importance of these residues to enzyme function.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)
Depositing User: Mr Matthew Proctor
Date Deposited: 25 Feb 2019 09:22
Last Modified: 25 Feb 2019 09:22
URI: http://etheses.whiterose.ac.uk/id/eprint/22964

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