Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium

Miller, Eric S.; Brenchley, Jean E.

doi:10.1128/jb.157.1.171-178.1984

Cited by 23 publications

(4 citation statements)

References 38 publications

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“…In the enteric coliform bacteria, ammonium can be assimilated by the production of glutamate from two reactions: glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH). It is thought that GOGAT functions when ammonium is limiting and that GDH functions when ammonium is present in concentrations greater than 1 mM (10,11,22,46,51). Glutamate can also be produced by the degradation of proline or arginine in Salmonella typhimurium (28,42).…”

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Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress

Botsford

Alvarez

Hernandez

et al. 1994

Appl Environ Microbiol

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Salmonella typhimurium accumulates glutamate in response to osmotic stress. Cells in aerobic exponential growth have an intracellular pool of approximately 125 nmol of glutamate mg of protein-'. When cells were grown in minimal medium with 500 mM NaCl, KCI, or sucrose, 290 to 430 nmol of glutamate was found to accumulate. Values were lower when cells were harvested in stationary phase. Cells were grown in conventional medium, harvested, washed, resuspended in the control medium or in medium with osmolytes, and aerated for 1 h. With aeration, glutamate was found to accumulate at levels comparable to those observed in exponential cultures. Antibiotics inhibiting protein synthesis did not affect glutamate accumulation when cells were aerated. Strains with mutations in glutamate synthase (git) or in glutamate dehydrogenase (gdh) accumulated nearly normal levels of glutamate under these conditions. A double (gdh glt) mutant accumulated much less glutamate (63.9 nmol mg of protein-'), but a 1.9-fold excess accumulated when cells were aerated with osmotic stress. Methionine sulfone, an inhibitor of glutamate synthase, did not prevent accumulation of glutamate in cells aerated with osmotic stress. Glutamate dehydrogenase is thought to have minimum activity when ammonium is limiting. Resuspending cells with limiting ammonium reduced glutamate production but did not eliminate accumulation of excess glutamate when cells were osmotically stressed. Amino oxyacetic acid, an inhibitor of transamination reactions, did not prevent accumulation of excess glutamate. The results indicate that neither glutamate dehydrogenase nor glutamate synthase is solely responsible for the excess glutamate that accumulates. It can be calculated that this excess glutamate accounts for only 1.0% of the cell's capacity to make glutamate. Very small changes in some of the reactions involved in glutamate metabolism could account for this.

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Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress

Botsford

Alvarez

Hernandez

et al. 1994

Appl Environ Microbiol

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“…The sequence of the enzyme (Bansal et al, 1989) exhibits considerable similarity, indicative of homology, with other NADP4-glutamate dehydrogenases, especially the enzyme from Escherichia coli (Valle etal., 1984; Blumenthaletal., 1975;Wootton et al, 1975). The gene encoding glutamate dehydrogenase from S. typhimurium has been cloned (Miller & Brenchley, 1984), its nucleotide sequence has been determined (Bansal et al, 1989) and wild-type as well as mutant enzymes have been expressed in E. coli and purified (Haeffner-Gormley et al, 1991). No three-dimensional structure of NADP4-glutamate dehydrogenase from any source has been reported.…”

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Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium

Haeffner‐Gormley¹,

Chen²,

Zalkin³

et al. 1992

Biochemistry

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Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Most microorganisms use an NADPH-dependent glutamate synthase (EC 2.6.1.53) to catalyze the conversion of a-ketoglutarate and glutamine to glutamate. In bacteria such as Salmonella typhimurium (4,24,31), Escherichia coli (1,26,33), and Klebsiella aerogenes (3,5,23), a second enzyme, glutamate dehydrogenase (EC 1.4.1.4), functions in the biosynthesis of glutamate from a-ketoglutarate and ammonia. Therefore, studying the regulation of these glutamate-forming enzymes is pivotal to understanding the overall nitrogen assimilatory process.…”

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Fine structure analysis of Salmonella typhimurium glutamate synthase genes

Madonna¹,

Fuchs²,

Brenchley³

1985

J Bacteriol

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Glutamate synthase activity is required for the growth of SalmoneUa typhimurium on media containing a growth-rate-limiting nitrogen source. Mutations that alter glutamate synthase activity had been identified in the glitB gene, but it was not known which of the two nonidentical subunits of the enzyme was altered. To examine the gene-protein relationship of the gil region, two nonsense mutations were identified and used to demonstrate that gitB encodes the large subunit of the enzyme. Six strains with independent Mu cts dl(lac bla) insertions were isolated, from which a collection of deletion mutations was obtained. The deletions were transduced with the nonsense mutations and 38 other gil point mutations to construct a fine-structure genetic map. Chromosome mobilization studies, mediated by Hfr derivatives of Mu cts dl lysogens, showed that gItB is transcribed in a clockwise direction, as shown in the S. typhimurium linkage map. Studies of the polar effects of three Mu cts dl insertions indicated that the gene for the small subunit maps clockwise to gltB and that the two genes are cotranscribed to form a gi operon.

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Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium

Cited by 23 publications

References 38 publications

Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress

Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress

Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium

Fine structure analysis of Salmonella typhimurium glutamate synthase genes

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