The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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