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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion. The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate. The crystals diffract well and X-ray photographs have established that they are in the space group R32. Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit. Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell. Such a conformational rearrangement may represent an important step in the catalytic cycle.
J Mol Biol 1992 Apr 20
PMID:Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum. Evidence for a ligand-induced conformational change. 134 42

The dinucleotide binding beta alpha beta motif in the crystal structures of seven different enzymes has been analysed in terms of their three-dimensional structures and primary sequences. We have identified that the hydrogen bonding of the adenine ribose to the glycine-rich turn containing the fingerprint sequence GXGXXG/A occurs via a direct or indirect mechanism, depending on the nature of the fingerprint sequence but independent of coenzyme specificity. The major determinant of the type of interaction is the nature of the residue occupying the last position of the above fingerprint. In the NAD(+)-linked dehydrogenases, an acidic residue is commonly used to form important hydrogen bonds to the adenine ribose hydroxyls and, hitherto, this residue has been thought to be an indicator of NAD+ specificity. However, on the basis of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum we have demonstrated that this residue is not a universal requirement for the construction of an NAD+ binding site. Furthermore, considerations of sequence homology unambiguously identify an equivalent acidic residue in both NADP+ and dual specificity glutamate dehydrogenases. The conservation of this residue in these enzymes, coupled to its close proximity to the 2' phosphate implied by the necessary similarity in three-dimensional structure to C. symbiosum GDH, implicates this residue in the recognition of the 2' phosphate either via water-mediated or direct hydrogen-bonding schemes. Analysis of the latter has led us to suggest that two patterns of recognition for the 2' phosphate group of NADP(+)-binding enzymes may exist, which are distinguished by the ionization state of the 2' phosphate.
J Mol Biol 1992 Nov 20
PMID:Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. 145 69

The Corynebacterium glutamicum gdh gene encoding NADP-dependent glutamate dehydrogenase (GDH) has been isolated by complementation of the Escherichia coli gdh mutant PA340. The gdh gene was subcloned into the E. coli/C. glutamicum shuttle vector pEK0 and introduced into C. glutamicum. Recombinant strains showed approximately eightfold higher specific GDH activity (15U mg protein-1) relative to the wild type (1.8U mg protein-1). Physiological studies with wild-type and recombinant C. glutamicum strains revealed no indication of significant regulation of gdh expression. The DNA sequence of 2082 bp, including the gdh gene, 5'-, and 3'-flanking regions, was determined. The structural gene consists of 1344 bp and codes for a polypeptide of 448 amino acid residues (Mr 49,152) showing up to 53.6% identity with reported amino acid sequences of glutamate dehydrogenases from other organisms. Northern blot hybridization revealed a 1.65kb mRNA transcript, indicating that the gdh gene of C. glutamicum is monocistronic. Transcription occurred from a G residue located 284 bp upstream of the AUG considered to be the translational initiation codon.
Mol Microbiol 1992 Feb
PMID:Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. 155 46

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.
Mol Cell Biol 1991 Sep
PMID:Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae. 165 57

NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent Kms for alpha-ketoglutarate, NADPH and NH4+ are 1.2 mM, 9.7 microM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 microM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 microM) yet N-ethylmaleimide does not. In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
Mol Cell Biochem 1991 Jun 26
PMID:Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum. 165 3

The NAC (nitrogen assimilation control) protein from Klebsiella aerogenes is a LysR-like regulator for transcription of several operons involved in nitrogen metabolism, and couples the transcription of these sigma 70-dependent operons to regulation by the sigma 54-dependent NTR system. NAC activates expression of operons (e.g. histidine utilization, hut), allowing use of poor nitrogen sources, and represses expression of operons (e.g. glutamate dehydrogenase, gdh) allowing assimilation of the preferred nitrogen source, ammonium. NAC is both necessary and sufficient to activate transcription, but the expression of the nac gene is totally dependent on the central nitrogen regulatory system (NTR) and RNA polymerase carrying the sigma 54 sigma factor (RNAP sigma 54). Nitrogen starvation signals the NTR system to transcribe nac, and NAC activates the transcription of hut, put (proline utilization), and urease. NAC does not affect the transcription of RNAP sigma 54-dependent operons like ginA or nifLA, which respond directly to the NTR system, but activates transcription of RNAP sigma 70-dependent operons. Thus NAC acts as a bridge between RNAP sigma 70-dependent operons like hut and the RNAP sigma 54-dependent NTR system. The activation of operons like hut by NAC in response to nitrogen starvation is at least superficially similar to their activation by CAP-cAMP in response to carbon and energy starvation.
Mol Microbiol 1991 Nov
PMID:The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes. 166 20

Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am-, acu- double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am+ (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3- mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.
Mol Gen Genet 1991 Oct
PMID:Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme. 168 13

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.
Mol Cell Biol 1991 Dec
PMID:Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae. 168 1

Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit.
Plant Mol Biol 1991 Nov
PMID:A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana. 171 78

We have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5' end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.
Mol Gen Genet 1991 Dec
PMID:The gene for a halophilic glutamate dehydrogenase: sequence, transcription analysis and phylogenetic implications. 176 32


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