The complete nucleotide sequence of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene

Kinnaird, Jane; Fincham, J. R. S.

doi:10.1016/0378-1119(83)90195-6

Cited by 174 publications

(79 citation statements)

References 19 publications

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“…The 24 residues which form the patch of high charge density in the Pf enzyme and which can be equivalenced across the entire enzyme family are highlighted in black boxes. References for the GluDH sequences given are : Clostridium symbiosum (Cs) [45]; Escherichia coli (Ec) [31]; Neurospora crassa (Nc) [46]; Clostridium difficile (Cd) [47]; Peptostreptococcus asaccharolyticus (Pa) [48]; Thermotoga maritima (Tm) [35]; Sulfolobus solfataricus (Ss) [36]; Thermococcus litoralis (Tl) [37], Pyrococcus endeavori (Pe) [38]; Pyrococcus furiosus (Pf) [49]. there are necessarily 26 residues that are unique to a single subcompared with the Pf enzyme.…”

Section: Sequence Alignment and Stability Ranking Ten Hexameric Resumentioning

confidence: 99%

Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family

Yip

Britton

Stillman

et al. 1998

European Journal of Biochemistry

116

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The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.Keywords : glutamate dehydrogenase; thermal stability; homology modeling; ion-pair network; Archaea.A proper understanding of the molecular basis of thermal ophiles has permitted direct structural comparisons, homologybased modelling studies and site-directed mutagenesis to be unstability in proteins could have important consequences for their application in a range of biotechnological processes. For exam-dertaken, combining sequence data on extremophilic enzymes with structural data from their mesophilic counterparts. Howple, the availability of enzymes with the appropriate specificity and capable of surviving for long periods at extreme temper-ever, to date, these analyses have not produced a consistent picture on the origins of thermal stability and suggestions put foratures could lead to the creation of novel applications of enzyme-based technology in industries such as those involved in ward to explain enhanced stability properties of proteins have included decreased flexibility of the protein [4Ϫ5] arising from the processing of paper, pulp or fibres [1Ϫ2]. The opportunities afforded by such applications have generated significant interest changes in interactions such as the increase in the number of ion-pairs [6Ϫ9], an increase in packing density [10], a decrease in the field and a large number of studies have been undertaken to unravel the molecular mechanisms involved in generating in the degree of cavity formation [11Ϫ12], a decrease in the sizes of loops linking secondary-structure elements [11], an thermostable enzymes. Recently, increasing attention has been focused on proteins from Archaea, a phylogenetically distinct increase in alanine residues located at the termini of surface A helices [13], a reduction in exposed surface area [14], an evolutionary kingdom that includes many extremophilic microorganisms and whose study has expanded our horizons on the increase in proline content [15Ϫ16], an increase in the number of disulphide bonds [17] and an increase in hydrophobic interaclimits of b...

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Section: Sequence Alignment and Stability Ranking Ten Hexameric Resumentioning

confidence: 99%

Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family

Yip

Britton

Stillman

et al. 1998

European Journal of Biochemistry

116

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“…That presumed d(ATG) start codon is preceeded by a short sequence d(CACA) similar to the start codon of other Neurospora crassa mRNAs [13,15,[22][23][24][25]. The start of the mRNA has not yet been determined.…”

Section: Isolation Of Cloned Cdma and Genomic Dnamentioning

confidence: 99%

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

1985

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The primary structure of the iron-sulfur subunit of ubiquinol -cytochrome c reductase from Neurosporu mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21 556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.Ubiquinol -cytochrome c reductase (cytochrome reductase) is a proton-translocating enzyme complex of the oxidative phosphorylation system in mitochondria. The enzyme isolated from Neurosporu consists of the cytochromes b and cl, an iron-sulfur subunit (Rieske 2Fe-2S protein, [l]) and six subunits without redox centers [2, 31.The three-dimensional structure of cytochrome reductase, the arrangement of the structure in the mitochondrial inner membrane and the topography of most of the subunits within the structure have been studied by electron microscopy of twodimensional crystals, neutron diffraction of enzyme/detergent preparations and biochemical characterization of isolated subunits [3 -71. With regard to the iron-sulfur subunit the studies showed that the subunit extends from the membrane with a large domain which carries the 2Fe-2S cluster into the intermembrane space of mitochondria and is anchored to the bilayer only by a small protein part.The import of the cytoplasmically synthesized subunits of the Neurospora cytochrome reductase (all except cytochrome b, which is a mitochondrial coded protein) into mitochondria has been studied recently [8]. The subunits, except the 14000-Da subunit, are synthesized as larger precursors and proteolytically processed during or after their import into mitochondria. Similar results were obtained for the yeast enzyme [9 -121.In this article we report on the primary structure of the iron-sulfur subunit of Neurospora cytochrome reductase in its

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“…Nearly 77% of the third-position choices are A's or T's, as would be expected for a sequence with this composition (28). Many of the codons are rarely used in strongly expressed E. coli genes (9) (for example, there are 28 GGA codons, 19 AGA codons, 7 CUA codons, and 5 AUA codons), yet a high level of expression was obtained in E. coli, in agreement with the observation (13) that protein elongation rates rarely put a severe limit on expression.…”

Section: Discussionmentioning

confidence: 99%

Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus

Snedecor¹,

Chu²,

Chen³

1991

J Bacteriol

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The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection in Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.As part of a program to develop live cell catalysts for the manufacture of amino acids by transatination of the corresponding alpha-keto acids, we cloned in Escherichia coli the gene for the NAD-dependent glutamate dehydrogenase of an anaerobic gram-positive coccus, Peptostreptococcus asaccharolyticus. This glutamate dehydrogenase is one of the few bacterial NAD-dependent glutamate dehydrogenases that have been purified and characterized (14,16,18,33). In P. asaccharolyticus, it acts in the hydroxyglutarate fermentation of glutamate, in which its normal role is the degradation of glutamate (15). We selected for the reverse of this reaction to obtain the clone. In this paper, we report the isolation, expression, and nucleotide sequence analysis of the P. asaccharolyticus gene for glutamate dehydrogenase. We compare the amino acid sequence of the translated gene with those of other glutamate dehydrogenases to show that this glutamate dehydrogenase is more closely related to the dual-specificity (NAD-and NADP-accepting) mammalian glutamate dehydrogenases than to the NADP-linked glutamate dehydrogenase of E. coli. MATERIALS AND METHODSBacterial strains. The strains used in this work are listed in Table 1. To construct strain Q100, we moved a host restriction mutation into a glutamate auxotroph by cotransduction with a nearby TnJO insertion. Strain PA340 was transduced with P1 phage prepared from strain LCK8, and the Tetr transductants were scored for the retention of the hsdR2 locus by comparing the efficiency of plaque formation when host-modified and unmodified P1 phages were plated on each transductant. Selection for the loss of the TnJO insertion was carried out by the method of Bochner and coworkers (2). DNA isolation. P. asaccharolyticus cells were grown anaerobically in Difco thiol broth. Cycloserine (6 ,ug/ml) was added just before the cells entered stationary phase in order to make them more susceptible to lysozyme action. The harvested cells were washed in 0.15 M NaCl and were then * Corresponding author. resuspended in 18.75% sucrose-37.5 mM Tris-HCl buffer (pH 8) containing 5 mg of lys...

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The complete nucleotide sequence of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene

Cited by 174 publications

References 19 publications

Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family

Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus

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