Bronagh M Hayden

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A GDH gene from Halobacterium salinarum has been cloned and sequenced and the publication assigns the sequence to the NADP+-glutamate dehydrogenase of this organism. We have expressed this gene in Escherichia coli and find that it encodes an NAD+-dependent glutamate dehydrogenase without activity towards NADP+. Further, peptide sequence from the two(More)
In earlier work, two glutamate dehydrogenase (GDH) proteins were purified from a strain of the halophilic archaeon Halobacterium salinarum (NRC-36014). One of these, an NAD+-specific enzyme, was matched to a cloned gene from H. salinarum (GenBank accession number: X63837 S75579) by sequencing peptide fragments. Analysis of enzymatic digests of the NADP+-GDH(More)
In vitro subunit hybridisation is a usehl technique for exploring inter-subunit interactions in oligomeric enzymes [I]. In order to explore the basis of allosteric bebaviour in glutamate dehydrogenase from Clmtridiurn syrnbimurn, hybrid hexamers of mutants, VIZ. triple mutant, K89L/A163G/S380A [2] and C320S [3] have been constructed. The mutant C320S is hUy(More)
The triple mutant K89L/A163G/S380A (inactive with glutamate but active with L-Nle and L-Met) and C320S (fully active with glutamate, entirely inactive with L-Nle and L-Met, and also lacking reactive cysteine) mutant of glutamate dehydrogenase (EC 1.4.1.2) of Clostridium symbiosum could be completely denatured by urea with the loss of structure and activity.(More)
In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both(More)
The catalytically disabled Asp165-->Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzyme in a standard glutamate oxidation assay and 1000-fold less activity in the reductive-amination reaction. The large reduction in the rate has been attributed to removal of the negative charge and the postulated(More)
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