BglF, the Escherichia coli beta-glucoside permease and sensor of the bgl system: domain requirements of the different catalytic activities.
The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of beta-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG. In the presence of beta-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports beta-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIAbgl and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl. Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i. e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl which occur upon the interaction of BglF with the sugar.
Chen
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Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Amster-Choder
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D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
C079638:BglF protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D001616:beta-Galactosidase