The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

Fincham, J. R. S.; Kinsey, John A.; Fuentes, Angel; Cummings, Nicola J.; Connerton, Ian F.

doi:10.1017/s0016672300004602

Cited by 7 publications

(5 citation statements)

References 19 publications

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“…Prior incubation of FIGDH with NADPH and 2-oxoglutarate abolished the lag in the forward initial velocity. Similar kinetic behavior was observed with a few am mutants of Neurospora crassa NADP-GDH [46]. Some of these were activated after pre-incubation with glutamate/succinate while others attained maximal reaction rate only after a lag period.…”

Section: Discussionsupporting

confidence: 78%

Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

2014

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NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical ‘one-way’ active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.

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Section: Discussionsupporting

confidence: 78%

Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

2014

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Section: Discussionmentioning

confidence: 88%

“…Complementation due to "stabilizing" mutant subunits was previously observed in the hexameric nicotinamide adenine dinucleotide phosphate-linked glutamic dehydrogenase (NADP-GDH) of Neurospora crassa (32). The mutant am 1 appeared to be conformationally stable but unable to bind NADP for catalysis (32), much like Q286R is conformationally stable but catalytically inactive. Initial investigation of mutant am 1 indicated that the mutant subunit complemented with a variety of other am mutants, and heterohexamers were active over a much wider range of conditions; that is, they were more stable than the homomeric form of the second am mutant (33).…”

Section: Discussionmentioning

confidence: 88%

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Mechanisms for Intragenic Complementation at the Human Argininosuccinate Lyase Locus

et al. 2001

Biochemistry

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Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.

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“…Mutants in A. nidulans gdhA, N. crassa am and S. cerevisiae GDH1 lacking NADP-GDH have reduced growth compared with wild type when ammonium is the sole nitrogen source (Arst and MacDonald, 1973;Kinghorn and Pateman, 1973;1975b;Fincham and Baron, 1977;Fincham, 1988;Avendano et al, 1997;Fincham et al, 2000;DeLuna et al, 2001). These genes are controlled by several conserved regulatory mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Dual DNA binding and coactivator functions of Aspergillus nidulans TamA, a Zn(II)2Cys6 transcription factor

Downes

Davis

Wong

et al. 2014

Molecular Microbiology

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SummaryTranscription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulans TamA and the related Saccharomyces cerevisiae Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function. TamA acts at several promoters as a coactivator of the global nitrogen GATA transcription factor AreA. We now show that TamA is the major transcriptional activator of gdhA, encoding the key nitrogen metabolism enzyme NADP-glutamate dehydrogenase. Moreover, activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. We show that the TamA DNA binding motif is required for DNA binding of FLAGepitope-tagged TamA to the gdhA promoter. We identify a conserved promoter element required for TamA activation, and show that TamA and AreA are reciprocally required for full binding at the gdhA promoter under conditions where AreA is inactive at most promoters but active at gdhA. Therefore TamA has dual functions as a DNA-binding transcription factor and a non-DNA-binding coactivator. Dual DNA-binding and coactivator functions provide an additional level of combinatorial control to mediate gene-specific expression.

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The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects – more mutants and a summary

Cited by 7 publications

References 19 publications

Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

Mechanisms for Intragenic Complementation at the Human Argininosuccinate Lyase Locus

Dual DNA binding and coactivator functions of Aspergillus nidulans TamA, a Zn(II)2Cys6 transcription factor

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