Functional studies of a glutamate dehydrogenase with known three-dimensional structure: steady-state kinetics of the forward and reverse reactions catalysed by the NAD+-dependent glutamate dehydrogenase of Clostridium symbiosum

Syed, Shabih E.-H.; Engel, Paul C.; Parker, David M.

doi:10.1016/0304-4165(91)90020-h

Cited by 56 publications

(65 citation statements)

References 45 publications

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“…Nitrate-grown A. niger mycelia were therefore suitable starting material in the purification of NADP-GDH activity. Dye-affinity chromatography has been exploited to purify NADP-GDH from a few organisms (Watson et al, 1978;Agrawal & Rao, 1983;Aguirre & Hansberg, 1988;Syed et al, 1991). Keeping this in mind, four different reactive dyes were explored and the Cibacron red LS-B-coupled matrix was most effective in purifying the A. niger NADP-GDH (Fig.…”

Section: Discussionmentioning

confidence: 99%

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Noor

Punekar

2005

Microbiology

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NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K m 34?7 mM) and ammonium (K m 1?05 mM; K i 0?4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K i values of 6?9, 9?2 and 202?0 mM. The A. niger enzyme showed allosteric properties and a sigmoid response (n H =2?5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.

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Section: Discussionmentioning

confidence: 99%

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Noor

Punekar

2005

Microbiology

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“…Although the overall similarities among these enzymes are not high, sequence similarities between these dehydrogenases clearly indicate the existence of an enzyme superfamily related by divergent evolution [18]. However, the substrate specificities of this superfamily are different; GluDH recognizes and binds glutamate in preference to all other amino acids [19], LeuDH and…”

Section: Methodsmentioning

confidence: 99%

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Kataoka

Tanizawa

2003

Journal of Molecular Catalysis B: Enzymatic

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The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward L-phenylalanine and phenylpyruvate with 1.6 and 7.8% of k cat values of the wild-type enzyme for the preferred substrates, L-leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.

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“…Depending on the carbon and nitrogen sources, different forms of GDH [NADP-, NAD-or NAD(P)-] have been reported from Clostridium symbiosum (Syed et al, 1991), Laccaria bicolor (Garnier et al, 1997), Escherichia coli (Maurizi & Rasulova, 2002), P. aeruginosa (Smits et al, 1984), Sulfolobus solfataricus (Schinkinger et al, 1991) and Salinibacter ruber (Bonete et al, 2003). The induction of NAD-, NADP-or NAD(P)-GDH has also been reported under varying environmental conditions such as salt (Bonete et al, 1990(Bonete et al, , 2003, temperature (Camardella et al, 2002) and nitrogen source (Abrahams & Abratt, 1998;Brown et al, 1973;LeJohn & McCrea, 1968).…”

Section: Discussionmentioning

confidence: 99%

Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4

Vamsee-Krishna

Phale

2008

Microbiology

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Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or a-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADPdependent GDH (NADP-GDH), while cells grown on glucose, 2¾ yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon sourcedependent presence of two (GDH I and GDH II ), three (GDH A , GDH B and GDH C ) and one (GDH P ) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.

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Functional studies of a glutamate dehydrogenase with known three-dimensional structure: steady-state kinetics of the forward and reverse reactions catalysed by the NAD+-dependent glutamate dehydrogenase of Clostridium symbiosum

Cited by 56 publications

References 45 publications

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4

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