Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601645
Title: The 2-oxoacid dehydrogenase multienzyme complex from thermophilic Archaea
Author: Marrott, Nia Laura
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2012
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Abstract:
The 2-oxoacid dehydrogenase multienzyme complexes (OADHCs) catalyse the following reaction: 2-Oxoacid + NAD+ + CoASH → Acyl-SCoA + CO2 + NADH + H+ These 5-10 MDa complexes comprise multiple copies of three enzymes: E1, E2, and E3. The structural core of the OADHC is usually composed of either 24 (cubic) or 60 (dodecahedral) E2 polypeptides, around which the E1 and E3 enzymes associate non-covalently. Due to a lack of detectable activity in cell extracts, it was originally thought that Archaea did not possess any OADHCs; instead they utilised the smaller ferredoxin oxidoreductase (FOR) family of enzymes. However, the OADHC genes have since been identified in an increasing number of aerobic archaea, and given that the genomes of these organisms have been streamlined to remove any unnecessary genes, these two observations suggest that archaea may utilise both FORs and OADHC. The Thermoplasma acidophilum OADHC component enzymes have been recombinantly expressed and assembled, resulting in a thermostable and thermoactive branched-chain OADHC. This complex provides a model to investigate stability, not only of the individual enzymes but also of a multi-protein complex and its assembly. The structure of this hyper-thermostable E2 core is crucial to the thermostability of the entire complex. The Tp. acidophilum E2 structure has been solved by analytical ultra-centrifugation, X-ray crystallography and small-angle X-ray scattering; these data have shown that this E2 assembles into a novel 42-mer structure, comprising 14 trimers, with both square and pentagonal faces, rather than either of the expected cubic 24-mer or dodecahedral 60-mer structures. Analysis of the Tp. acidophilum E2 structure has identified an isoleucine ‘anchor’ residue that is key to the formation of the trimer-trimer interactions. The isoleucine side-chain extends from the C-terminal helix of one trimer into a hydrophobic pocket of the adjacent trimer and vice versa. The putative anchor residue has been removed by the insertion of a premature stop codon, resulting in E2 trimers that no longer assemble into a 42mer. This mutation has no affect on the tertiary structure of the trimers or its acyltransferase activity; moreover, the trimeric E2 retains the ability to bind the E1 and E3 enzymes, resulting in the first example of a functional trimeric OADHC.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.601645  DOI: Not available
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