Energy generating processes like the citric acid cycle are a pivotal part of metabolism. Members of the 2-oxoacid dehydrogenase multienzyme complex (OADHC) superfamily feed into and act within the citric acid cycle. OADHCs are composed of three enzymes: 2-oxoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Covalent attachment of lipoic acid (LA) to E2 is essential for overall OADHC activity. Although thought to be absent in Archaea, it has recently been found that Thermoplasma acidophilum has all the components for an active recombinant OADHC (Heath et al., 2007). Recent studies have further suggested that Tp. acidophilum may have an enzyme to covalently attach LA to E2 (Sun et al., 2007; McManus et al., 2006). This work describes the cloning and recombinant expression of the Thermoplasma lipoate protein ligase (Tp. LplA), its C-terminal domain and a fusion protein composed of the above two proteins. Both proteins are required for lipoylation of E2 in vitro. For the first time, in vivo lipoylation of E2 in Tp. acidophilum cell cultures is also being reported. The effect of lipoylation and temperature on the Thermoplasma OADHC assembly has also been studied. This study revealed the temperature dependence of the E2 core and the whole complex assembly. These findings are in line with the optimum growth temperature of Tp. acidophilum. Dynamic light scattering and analytical ultracentrifugation were used to determine the molecular mass of whole OADHC. The molecular mass was determined to be 5 MDa with an octahedral geometry of the E2 core. The results of this work strengthen the assumption that these enzyme systems may have had or potentially have a role in the Archaea. This may hold further clues to the evolutionary relationship between the three kingdoms of life and the role of OADHCs/lipoylation in the Archaea. The temperature dependent assembly of the complex and thermostability of these proteins may also provide a model to study thermostability and protein-protein interactions at high temperatures.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:503364 |
| Date | January 2009 |
| Creators | Posner, Mareike |
| Contributors | Danson, Michael ; Hough, David |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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