Monika Schneider

• First described in the bacterium Pseudomonas saccharophila the Entner-Doudoroff- pathway (ED-pathway) is one of the three most widespread metabolic pathways for glucose and other energy sources. The other two pathways are the Embden-Mayerhof- Parnas-pathway (Glycolysis) and the Pentosephosphate-pathway. For Zymomonas mo bilis (also known as the ”yeast of tequila“) the ED-pathway is the only way to generate energy while synthesizing ethanol as a byproduct. Additionally, the ED-pathway is used by many pathogens, making it a potential target for antibiotics. Its high flux rate makes it interesting for biotechnological applications. Still there is not much known about the ED-pathway to this day. The main reason for this is probably the instability of its key en zyme: the Entner-Doudoroff dehydratase (6-phosphogluconate dehydratase, EDD). This makes the research on EDD the fulcrum of understanding the whole ED-pathway. In this work bioinformatic analyses on the distribution of EDD in bacteria show that mostly gammaproteobacteria, actinobacteria, alphaproteobacteria and betaproteobac teria have an EDD. An alignment of more than 200 sequences made it possible to distinguish EDD from other members of the IlvD(2,3-Dihydroxy-3-methyl/Isovalerate dehydratase)/EDD-family. It also shows two highly conserved regions. For the EDD from Thermomonospora curvata those are 165CDKxxP170 and 521DxRxSG526. Characterization of the heterologously expressed and affinity-tagged isolated EDDs of E. coli, Z. mobilis and T. curvata was carried out. For the latter thermostability and stability of the activity over 7 weeks in an anaerobic environment was demonstrated, properties which are for many applications of key interest. All three have a [4Fe-4S]- cluster in the active center, not a [2Fe-2S]-Cluster. Kinetic data has been determined by development of a coupled activity assay. Mutagenesis followed by characterization of the mutants shows the coordination of the [4Fe-4S]-cluster of the enzyme from T. curvata by C123, C165 and C232, leaving one ”corner“ of the cluster for the coordination of the substrate, as described for aconitase. This iron-ion could be substituted selectively by 57Fe to carry out site-specific Mößbauer spectroscopy. For the first time Mößbauer spectroscopy and Resonance Raman spectroscopy have been carried out for an EDD. Together with EPR spectroscopy information on the active center was obtained. A successful synthesis of the substrate 6-phosphogluconate from glucose gave the opportunity to look into the coordination of the substrate. Furthermore, it enabled the synthesis of 13C labeled substrate. The determination of the mechanism was supported by NMR measurements, both time dependent (1H) and 2-dimensional (COSY, NOESY). The hypothesis that the ketonisation is not catalysed by the EDD was disproven. Both in the forward and the reverse reaction the pro-S position at C-3 was selectively deuterated by deuterium oxide.