Genetic and biochemical analysis of phosphatase activity of Escherichia coli NRII (NtrB) and its regulation by the PII signal transduction protein.
Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.
Pioszak
Augen A
AA
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Ninfa
Alexander J
AJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D051922:PII Nitrogen Regulatory Proteins; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D051922:PII Nitrogen Regulatory Proteins; C404143:PIID regulatory protein, Bacteria; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases