|dc.description.abstracteng||In most organisms the tricarboxylic acid (TCA) cycle is a fundamental hub in metabolism. The TCA cycle is the origin and the endpoint of a broad spectrum of intermediates and provides the cell with energy and cellular building blocks. Especially in bacteria the pathway importantly contributes to their broad metabolic abilities.
In this work it is shown that the majority of the TCA cycle enzymes in Bacillus subtilis interact with each other via protein-protein interactions and a model for a TCA cycle metabolon is established. The assembly in complexes seems to be a common feature of metabolic enzymes like the enzymes of the TCA cycle. A complex of enzymes might lead to higher local substrate concentrations and might enhance metabolic fluxes by substrate channeling. Furthermore, interactions between enzymes of the TCA cycle and enzymes of connected metabolic pathways, like gluconeogenesis or nitrogen metabolism, are identified. Consequently, it can be assumed that the connection of the pathways via protein-protein interactions might directly trigger the flux through the different pathways.
Another aim of this work was to elucidate the mechanism of malate-mediated carbon catabolite repression (CCR) and to analyze the role of the B. subtilis malate dehydrogenases in malate metabolism. For malate-mediated CCR an unknown mechanism was supposed to be active in B. subtilis. However, in this work it is demonstrated that malate-mediated repression is achieved by the global mechanism of CCR via the CcpA/HPrK pathway and that malate triggers the formation of ATP and fructose 1,6-bisphosphate levels sufficient for CcpA/HPrK pathway activation. Malate is a preferred carbon source of B. subtilis and for an efficient utilization B. subtilis relies on a number of specific transporters and enzymes. Here, the essential role of the malate dehydrogenase and the phosphoenolpyruvate carboxykinase in malate utilization and the role of the malic enzymes of B. subtilis to supply the cell with NADPH, NADH and ATP are demonstrated.
Finally a novel mechanism of TCA cycle branch control is identified. In B. subtilis, the regulation of the TCA cycle is mainly exerted at the transcriptional level. Especially the expression of the first two enzymes, citrate synthase and aconitase, is controlled by a complex interplay of several transcription factors. In this work it is demonstrated that the iron regulatory protein aconitase is able to bind the mRNA of citrate synthase under high intracellular citrate concentrations and thus to destabilize its transcript to reduce the level of citrate synthase protein in the cell.||de