PMID:11133942

Citation	Lu, CD and Abdelal, AT (2001) The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation. J. Bacteriol. 183:490-9
Abstract	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.
Links	PubMed PMC94904 Online version:10.1128/JB.183.2.490-499.2001
Keywords	Allosteric Regulation; Amino Acid Sequence; Arginine/pharmacology; Base Sequence; Citric Acid/pharmacology; Cloning, Molecular; Enzyme Induction; Gene Expression Regulation, Bacterial; Glutamate Dehydrogenase/drug effects; Glutamate Dehydrogenase/genetics; Glutamic Acid/pharmacology; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Mutation; NAD; Promoter Regions, Genetic; Protein Conformation; Pseudomonas aeruginosa/enzymology; Pseudomonas aeruginosa/genetics; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEAE:DHE2		GO:0004352: glutamate dehydrogenase (NAD+) activity	ECO:0000314:		F		Table 2 showed that NAD-GDH was induced 27-fold when PAO1 was grown with arginine as the sole source of carbon and nitrogen relative to the level found when grown with succinate and ammonia as carbon and nitrogen sources, respectively. Proving that the gdhB gene encodes an arginine-inducible NAD+ dependent glutamate dehydrogenase.	complete CACAO 7655
PSEAE:DHE2	enables	GO:0004352: glutamate dehydrogenase (NAD+) activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

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