Feedback-resistant mutations in Bacillus subtilis glutamine synthetase are clustered in the active site.
The feedback-inhibited form of Bacillus subtilis glutamine synthetase regulates the activity of the TnrA transcription factor through a protein-protein interaction that prevents TnrA from binding to DNA. Five mutants containing feedback-resistant glutamine synthetases (E65G, S66P, M68I, H195Y, and P318S) were isolated by screening for colonies capable of cross-feeding Gln(-) cells. In vitro enzymatic assays revealed that the mutant enzymes had increased resistance to inhibition by glutamine, AMP, and methionine sulfoximine. The mutant proteins had a variety of enzymatic alterations that included changes in the levels of enzymatic activity and in substrate K(m) values. Constitutive expression of TnrA- and GlnR-regulated genes was seen in all five mutants. In gel mobility shift assays, the E65G and S66P enzymes were unable to inhibit TnrA DNA binding, while the other three mutant proteins (M68I, H195Y, and P318S) showed partial inhibition of TnrA DNA binding. A homology model of B. subtilis glutamine synthetase revealed that the five mutated amino acid residues are located in the enzyme active site. These observations are consistent with the hypothesis that glutamine and AMP bind at the active site to bring about feedback inhibition of glutamine synthetase.
Fisher
Susan H
SH
Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA. shfisher@bu.edu
Wray
Lewis V
LV
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005974:Glutamate-Ammonia Ligase