In den Firmicutes wird der Induktorausschluss (Katabolitrepression) durch das am Serin46 phosphorylierte HPr (PTS) vermittelt. Der genaue Mechanismus war jedoch unklar. Um diese Frage auf der Grundlage von isolierten Proteinen zu klären, wurde ein zum Escherichia coli Maltose-/Maltodextrin-ABC-Transporter homologes System aus Lactobacillus casei BL23 (MalE1-MalF1G1K12) als Modellsystem genutzt. Im Rahmen der Promotion wurde über isothermale Titrationskalorimetrie und Fluoreszenzspektroskopie gezeigt, dass das Bindeprotein MalE1 lineare und zyklische Maltodextrine, aber keine Maltose bindet. Experimentell ermittelte dreidimensionale Strukturen von MalE1 im Komplex mit diesen Zuckern belegten eine vergleichbar geschlossene Konformation und dienten zusätzlich als Grundlage, um die fehlende Maltosebindung zu erklären. Die Stimulierung der ATPaseaktivität des in Liposomen und Nanodiscs eingebauten Komplexes wurde jedoch hauptsächlich durch eine MalE1-Beladung mit linearen Maltodextrinen bewirkt. Eine bis zu 85 %ige Inhibierung der ATPaseaktivität durch P-Ser46-HPr belegte erstmals in vitro eine Interaktion von mehr als einem phosphorylierten Protein mit dem Transporter. Analog zum EIIAGlc-Inhibitor des homologen Systems aus E. coli wurden über Quervernetzungsexperimente und massenspektrometrische Analysen Interaktionen mit dem MalK1-Dimer als interagierende Komplexeinheit in der Nähe des Walker A-Motivs nachgewiesen. Über Fluoreszenzmessungen in Anwesenheit des ATP-Analogons TNP-ATP wurde eine unbeeinflusste ATP-Bindung und damit eine fehlende Blockade der γ-Phosphatbindestelle des Walker-A Motivs durch die Phosphorylgruppe von P-Ser46-HPr bestimmt. Die folgende Substitution verschiedener positiv geladener MalK1-Reste, die als potenzielle Interaktionsstellen für die Phosphorylgruppe fungieren könnten, identifizierte K63 in der Nähe des Walker A-Motivs als ersten möglichen Partner. Der genaue Mechanismus der Inhibierung bleibt jedoch unklar. / Catabolite repression is a global mechanism which controls the utilization of carbohydrates in bacteria. In Firmicutes HPr, a component of the phosphoenolpyruvate carbohydrate phosphotransferase system, prevents the uptake of less preferred sugars but only when it is phosphorylated at serine46. However the exact mechanism was unclear. To address this question the purified ATP-binding cassette transporter from Lactobacillus casei BL23 (MalE1-MalF1G1K12) was used as a model system, which is homologous to the Escherichia coli maltose/maltodextrin ABC importer. Isothermal titration calorimetry and fluorescence spectroscopy revealed that the binding protein MalE1 binds linear and cyclic maltodextrins but not maltose. Experimentally determined three-dimensional structures from MalE1 in complex with these sugars show a comparably closed conformation and served as a basis to explain the lack of maltose binding. The stimulation of the ATPase activity of the transporter incorporated in liposomes and nanodiscs however, was mainly caused by MalE1 loaded with linear maltodextrins. For the first time an inhibition of ATPase activity by P-Ser46-HPr up to 85 % and an interaction of more than one phosphorylated protein with the transporter was demonstrated. Analogous to the EIIAGlc inhibitor of the homologous system from E. coli, cross-linking experiments and mass spectrometric analyzes revealed interactions with the MalK1 dimer near the Walker A motif. Fluorescence measurements in the presence of the ATP analogue TNP-ATP, however, revealed an unaffected ATP binding and thus a lack of blockade of the γ-phosphate binding site (Walker A motif) by the phosphoryl group from P-Ser46-HPr. The following substitution of several positively charged MalK1 residues that could act as potential sites of interaction for the phosphoryl group, identified K63 near the Walker A motif as the first potential partner. The exact mechanism of inhibition, however, remains unclear.

Inositol dehydrogenase (IDH) catalyzes the oxidation of myo-inositol to scyllo-inosose using NAD+ as the coenzyme. IDH-related genes (Lp_iolG1 to Lp_iolG4) from Lactobacillus plantarum WCSF1 and (Lc_iolG1 and Lc_iolG2) from Lactobacillus casei BL23 were cloned into the vector pQE-80L, expressed in E. coli host cells and the proteins were purified to homogeneity. IDH activity of the purified enzymes was explored with myo-inositol and other structurally related compounds. It was found that IDH-related enzymes from L. plantarum WCSF1 did not exhibit any activity with tested substrates but, LcIDH1 and LcIDH2 from L. casei BL23 showed activity with myo-inositol and other related compounds. pH-rate profile studies have demonstrated the optimum pH for the reactions catalyzed by the active enzymes. Steady-state kinetics of the active enzymes was performed as with IDH from Bacillus subtilis (BsIDH), revealing that LcIDH1 is a myo-inositol dehydrogenase and LcIDH2 is a scyllo-inositol dehydrogenase. Both LcIDH1 and LcIDH2 are observed to be NAD+-dependent. Kinetic isotopic effect experiments for LcIDH1 have demonstrated that the chemical step in the reaction is partly rate-limiting. Substrate spectrum of LcIDH1 and LcIDH2 was explored and compared to BsIDH. Finally, a multiple sequence alignment of IDH-related enzymes was performed and the proposed consensus sequence motifs were considered to understand the activity differences between these enzymes.