Functional roles of the conserved Glu304 loop of Bacillus subtilis glutamine synthetase.
The enzymatic activity of Bacillus subtilis glutamine synthetase (GS), which catalyzes the synthesis of glutamine from ammonium and glutamate, is regulated by glutamine feedback inhibition. The feedback-inhibited form of B. subtilis GS regulates the DNA-binding activities of the TnrA and GlnR nitrogen transcriptional factors. Bacterial GS proteins contain a flexible seven-residue loop, the Glu304 flap, that closes over the glutamate entrance to the active site. Amino acid substitutions in Glu304 flap residues were examined for their effects on gene regulation, enzymatic activity, and feedback inhibition. Substitutions in five of the Glu304 loop residues resulted in constitutive expression of both TnrA- and GlnR-regulated genes, indicating that this flap is important for regulating the activity of these transcription factors. The residues in the highly conserved Glu304 flap appear to be optimized for glutamate binding because mutant enzymes with substitutions in five of the flap residues had increased glutamate Km values compared to that for wild-type GS. The E304A and E304D substitutions increased the ammonium Km values compared to that for wild-type GS and conferred high-level resistance to inhibition by glutamine, glycine, and methionine sulfoximine. A model for the role of the Glu304 residue in glutamine feedback inhibition is proposed.
Wray
Lewis V
LV
Department of Microbiology, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118-2526, USA.
Fisher
Susan H
SH
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D012097:Repressor Proteins; C101458:ScgR protein, Bacillus subtilis; D005973:Glutamine; D005974:Glutamate-Ammonia Ligase