Effect of mutations in Escherichia coli glnL (ntrB), encoding nitrogen regulator II (NRII or NtrB), on the phosphatase activity involved in bacterial nitrogen regulation.
We examined the effects of mutations in glnL, encoding the signal-transducing kinase/phosphatase nitrogen regulator II (NRII), on the regulated phosphatase activity involved in nitrogen regulation. With wild-type NRII, this phosphatase activity was only observed in the presence of the signal transduction protein II (PII). Three different glnL mutations result in altered NRII proteins that had phosphatase activity in the absence of PII. The most active of these contained an alteration of the site of NRII autophosphorylation, histidine 139, to asparagine (H139N). The phosphatase activity of the NRII-H139N protein was further stimulated by the PII protein and by ATP. This suggests that the PII protein is not directly involved in a catalytic step of the regulated phosphatase activity but rather plays a regulatory role. We also measured the effect on the regulated phosphatase activity of alterations at conserved residues in the kinase/phosphatase domain of NRII and the effect of deleting the non-conserved N-terminal domain of NRII. For this we used fusion proteins containing the Escherichia coli maltose-binding protein (MBP) linked to the protein of interest. A protein consisting of MBP linked to wild-type NRII was a less active kinase than was wild-type NRII but in the presence of PII had wild-type phosphatase activity. A protein consisting of MBP linked to just the C-terminal domain of wild-type NRII had kinase activity but lacked phosphatase activity. Alterations at the highly conserved residues Asp-287, Gly-289, and Gly-291 in NRII affected both activities. A fusion of MBP to the NRII-H139N protein lacked kinase activity but had phosphatase activity in the absence of PII. Thus, while the kinase and phosphatase activities of NRII could be genetically separated, some of the highly conserved residues in the C-terminal domain of NRII (Asp-287, Gly-289, Gly-291) are apparently important for both activities.
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
E S
ES
Kamberov
M R
MR
Atkinson
P
P
Chandran
A J
AJ
Ninfa
D000595:Amino Acid Sequence; D001216:Asparagine; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; C404143:PIID regulatory protein, Bacteria; D001216:Asparagine; D010744:Phosphoric Monoester Hydrolases; D009584:Nitrogen