title: Biochemical characterization of a nitrogen-type phosphotransferase system reveals that enzyme EI(Ntr) integrates carbon and nitrogen signaling in Sinorhizobium meliloti.
abstract: In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTS(Ntr)). In this PTS(Ntr), the protein HPr is phosphorylated on histidine-22 by the enzyme EI(Ntr) and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EI(Ntr), both proteins were purified and the activity of EI(Ntr) was measured. Experimentally determined kinetic parameters of EI(Ntr) activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EI(Ntr). Binding experiments using the isolated GAF domain of EI(Ntr) (EIGAF) showed that it is the domain responsible for detection of glutamine. EI(Ntr) activity was not affected by α-ketoglutarate, and no binding between the EIGAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EI(Ntr) phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine).
authors.affiliation: University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut, USA.
authors.forename: Reed A
authors.initials: RA
authors.lastname: Goodwin
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Gage
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005973:Glutamine; D007656:Ketoglutaric Acids; D009584:Nitrogen / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D005973:Glutamine; D002244:Carbon; D010770:Phosphotransferases; D009584:Nitrogen