Cloning and characterisation of the plant pyruvate dehydrogenase complex components

McGow, Donna (2002) Cloning and characterisation of the plant pyruvate dehydrogenase complex components. PhD thesis, University of Glasgow.

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Abstract

Multienzyme complexes are non-covalent assemblies composed of one or more copies of their constituent enzymes that act cooperatively to catalyse a linked series of reactions. These multienzymes complexes are of great interest in the study of protein-protein interactions, substrate channelling and the catalytic and regulatory advantages conferred by their organisation into multimeric complexes. One such complex is pyruvate dehydrogenase complex (PDC), the principal member of the 2-oxo acid dehydrogenase complex family. PDC is located in the mitochondrial matrix in eukaryotes, where it catalyses the irreversible oxidative decarboxylation of pyruvate committing its acetyl-CoA product to enter into the citric acid cycle or ketone body formation. Plants are unique in having two distinct and spatially separate complexes; a mitochondrial complex similar to the mammalian and a chloroplastic complex with a unique anabolic function in providing acetyl-CoA and NADH for de novo fatty acid synthesis. Although much is known about the organisation and stoichiometry of the PDC from mammalian, yeast and prokaryotic sources little is known of the composition, subunit organisation and stoichiometry of the plant equivalent. Until recently, studies have been hindered by the low abundance of PDC in plant tissues. Nevertheless the plant PDC from both organelles are thought to be composed of the same three enzymes found in mammalian PDC designated E1, E2 and E3. In all PDCs studied to date E1 and E3 are arranged around an oligomeric E2 core. Two types of E2 core have been identified; the 24meric cube displaying octahedral (432) symmetry and the 60meric pentagonal dodecahedron displaying icosahedral (532) symmetry. In this thesis, various enzymes from plant mitochondrial and chloroplastic PDCs have been cloned from either recombinant plasmids containing the genes encoding PDC components or from cDNA produced from mRNA. These enzymes were cloned into different expression vectors that incorporate expression tags (His-tag or GST-fusion) to the N-terminal region of the recombinant enzymes and successfully expressed in E. coli. Subsequently these enzymes were purified to near homogeneity by Zn2+ affinity chromatography, as judged by Coomassie blue staining. High yields of enzymatically active recombinant enzymes were achieved using this purification method. The plant mitochondrial and chloroplastic E2 component was expressed as either the mature E2 enzyme or as a truncated form comprising the lipoyl domain(s) and adjacent subunit (E1 and/or E3) binding domain. Successful lipoylation of E2 indicates that these recombinant enzymes were capable of folding into their native 3D structures. A monoclonal antibody (mAb) specific for lipoylated human E3-BP and E2 was used to detect lipoylated plant E2s. However, this antibody only recognised the mitochondrial E2. The specificity of the antibody for mitochondrial E2s suggests that in addition to the presence of the lipoic acid cofactor the antibody also recognises regions of sequence specific to the mitochondrial E2 lipoyl domain. Sequence alignments of mitochondrial and chloroplastic/ plastidic lipoyl domains reveal differences that may be the potential epitope(s) for the mAb. Lipoylation of the chloroplastic E2 was verified by measuring the mass of the E2 didomain GST-fusion proteins by MALDI-TOF after expression in media with or without exogenous lipoic acid. The probable subimit organisation of plant mitochondrial and chloroplastic E2 cores was investigated by analysing their Mr values using analytical ultracentrifugation in the sedimentation equilibrium mode. These preliminary studies show a distinct difference in Mr value between the two E2 complexes. The chloroplastic E2 was estimated to have a Mr value consistent with it assembling into a 24meric oligomer while the Mr value estimated for the mitochondrial E2 suggests assembly into a 60meric core. Finally, the expression of recombinant potato E3 and pea L-protein in E. coli did not hinder these enzymes from forming their native dimeric structure. The stability of the dimeric structure was studied and was proved to be essential for E3 activity, with loss of secondary and tertiary structure correlating with the loss of activity. Fluorescence studies on both the potato E3 and pea L-protein showed the release of FAD with the unfolding of the E3 enzyme and is consistent with the loss of E3 activity. The effect of the 31 amino acid mitochondrial targeting sequence on the structure of the L-protein was also studied using circular dichroism and fluorescence. The in vivo function of a targeting peptide is to maintain the protein in a transient unfolded form until the protein reaches its final destination with the cell. The L-protein precursor was expressed in E. coli in a partially unfolded form possibly similar to that found in vivo. This was further substantiated by its inactivity and insolublity and by the structural changes observed by CD and fluorescence after pre-treatment with urea.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J G Lindsay
Keywords: Plant sciences
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-72760
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: https://theses.gla.ac.uk/id/eprint/72760

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