This title appears in the Scientific Report :
2011
Der Oxoglutarat-Dehydrogenase-Komplex in $\textit{Corynebacterium glutamicum}$ und seine Interaktion mit Odhl
Der Oxoglutarat-Dehydrogenase-Komplex in $\textit{Corynebacterium glutamicum}$ und seine Interaktion mit Odhl
In the glutamate-producing bacterium $\textit{Corynebacterium glutamicum}$, the oxoglutarate dehydrogenase (ODH) is part of a mixed pyruvate/oxoglutarate dehydrogenase complex in which the peptide OdhA has E1 and E2 activity with oxoglutarate as substrate. The activity of the ODH is modulated by the...
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Personal Name(s): | Raasch, Katharina (Corresponding author) |
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Contributing Institute: |
Biotechnologie 1; IBT-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2012
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Physical Description: |
VI, 86 S. |
Dissertation Note: |
Universität Düsseldorf, Diss., 2011 |
ISBN: |
978-3-98336-764-1 |
Document Type: |
Dissertation / PhD Thesis |
Research Program: |
Biotechnologie |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health
48 |
Subject (ZB): | |
Publikationsportal JuSER |
In the glutamate-producing bacterium $\textit{Corynebacterium glutamicum}$, the oxoglutarate dehydrogenase (ODH) is part of a mixed pyruvate/oxoglutarate dehydrogenase complex in which the peptide OdhA has E1 and E2 activity with oxoglutarate as substrate. The activity of the ODH is modulated by the regulatory protein OdhI which can be reversibly phosphorylated. In the unphosphorylated state, OdhI binds to OdhA and inhibits its activity. The phosphorylation of Thr-14 in OdhI, by the specific protein kinase PknG, leads to an intramolecular interaction between the phospho-threonin residue 14 (pThr-14) and the carboxyterminal FHA domain in OdhI. As a consequence, OdhI interaction with OdhA is no longer possible and OdhA remains active. This regulation of the ODH activity by posttranslational modification of OdhI is so far an unknown mechanism. Hence, the present study was aiming for a detailed analysis on the interaction between OdhI and OdhA, yielding the following results: (1) AceF possesses three lipoyl domains. The lipoyl domain L1 of AceF has a considerably lower accessibility to the active sites of the PDH/ODH complex compared to the L3 domain. In particular, the accessibility is lower for the active sites required for ODH activity. (2) Two aminoterminal truncated derivatives OdhA$\Delta$89 and OdhA$\Delta$354 showed undiminished E1 activity in comparison to native OdhA (1221 aa). In contrast, OdhA$\Delta$395 was catalytically inactive. Nevertheless, all three OdhA derivatives still showed unchanged interaction with OdhI. (3) The FHA domain of OdhI was sufficient for the complete inhibition of the ODH activity. Analysis of mutations revealed that the highly conserved SXXH motif in the FHA domain, essential for the binding of pThr-14, is apparently not involved in the protein-protein interaction with OdhA. (4) A Cys-704-Arg OdhA mutein was identified by an $\textit{in vivo}$ selection system, with the mutein showing a 50 % reduced interaction with OdhI. Hence Cys-704 of OdhA considerably affects interaction with OdhI. (5) X-ray structure analysis, chemical cross-linking, as well as $\textit{in silico}$ modeling of OdhA and OdhI suggests a spatial proximity of Glu-472 (OdhA) and Asp-126 (OdhI) in the OdhA/OdhI complex. (6) Mutations of Tyr-111 and Arg-117 in OdhI led to a complete loss of interaction with OdhA, and are thus likely to be directly involved in protein-protein interaction. Taken together, interaction sites between OdhA and OdhI were further confined and in both proteins single amino acid residues were identified, thus contributing to the further understanding of OdhA/OdhI interaction complex. |