Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Nitrate reductase (NR) assays revealed a bispecific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%-77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch.
Mol Gen Genet 1991 May
PMID:Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions. 167 24

The nifH1 gene of Methanococcus thermolithotrophicus, which encodes the putative dinitrogenase reductase of an archaeon, was accurately transcribed in a homologous cell-free transcription system. Extracts of cells grown with N2 or ammonia as nitrogen source initiated transcription at the nifH1 promoter with similar efficiencies. We confirmed that cells grown under non-N2-fixing conditions do not contain significant amounts of nifH1-specific mRNA. The levels of cell-free transcription initiation at the nifH1 promoter were similar to those observed at a tRNA promoter. The DNA sequence from -40 to +5 relative to the initiator nucleotide of nifH1 mRNA contained all the information required for promoter activity. A mutational analysis of this section of DNA demonstrated that a TATA box at -25 and the TTGT motif (initiator element) at the transcription start site are essential for cell-free transcription. These elements are similar to the structural determinants of a known tRNA promoter of Methanococcus. Mutation of a sequence, showing homology to the bacterial NifA site, which overlaps the transcription start site, did not affect promoter activity. Hence, cell-free transcription of the Methanococcus nifH1 gene is independent of upstream activator elements and does not require alternate cis-acting sequences that differ from the methanogen consensus promoter. These findings suggest that the activation of nif promoters is brought about by fundamentally different mechanisms in Archaea and bacteria.
Mol Gen Genet 1992 Jan
PMID:Cell-free transcription of the nifH1 gene of Methanococcus thermolithotrophicus indicates that promoters of archaeal nif genes share basic features with the methanogen consensus promoter. 173 98

The cloning of the positive regulatory gene, uaY, which mediates uric acid induction of enzymes and permeases of the purine degradation pathway in the fungus Aspergillus nidulans is described here. The 4 kb uaY transcript is constitutively synthesised, it is not repressed by ammonia and its transcription does not require the AreA wide-domain transcription factor. We have determined that four deletions, which have been genetically characterised, are confined to a segment of 0.9 kb. Two other deletions are double events; each is a deletion of about 1 kb plus an insertion. The positions of the deletions confine 9 out of the 11 mapped putative point mutations within a 1 kb segment. Two other non-revertible alleles, which mapped as point mutations, are insertions of at least 11 and 18 kb respectively. The pattern of gene conversion within the uaY gene was described previously. The results reported here demonstrate that conversion of sequences of at least 18 kb can occur in A. nidulans.
Mol Gen Genet 1991 Dec
PMID:Molecular cloning of the uaY regulatory gene of Aspergillus nidulans reveals a favoured region for DNA insertions. 176 35

A gene bank of Azospirillum lipoferum Br17 constructed in the vector lambda GEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA-like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA-homology region were found. Mutants with Nif- phenotype were obtained by site-directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA-like gene. Construction of a nifA-lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA-like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.
Mol Microbiol 1991 Nov
PMID:Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia. 177 63

2,4-Dialkyl-5(4H)-oxazolones are well-recognized intermediates in some aminolysis reactions in peptide synthesis. Using the MOPAC molecular orbital programs, detailed geometric and energetic characteristics of the elementary reaction pathways for the additions of water and ammonia to 2-methyl-5(4H)-oxazolone have been determined at the AM1 level. The results demonstrate that the additions must be parsed into a two-step mechanism involving formation of the alpha-hydroxyimine followed by tautomerization to the parent N-acetylamino acid or amide.
J Comput Aided Mol Des 1991 Dec
PMID:Reaction mechanisms in peptide synthesis. Part 1. Semiquantitative characteristics of the reactivity of 2-methyl-5(4H)-oxazolone with water and ammonia in the gas phase and weakly polar media. 181 92

We had concluded in previous work that ring opening of a 2-alkyl-5(4H)-oxazolone by water or ammonia leads to transient high-energy imidol intermediates which instantly tautomerize to the native amides. Using the MOPAC molecular orbital program, detailed geometric and energetic characteristics of the tautomerism of a peptide bond have been determined on the AM1 level. The results demonstrate that tautomerism of a peptide bond comprises a three-stage process involving three successive transition states and a bimolecular mechanism: (i) E----Z peptide bond isomerization followed by dimerization, (ii) concerted double-hydrogen exchange leading to an alpha-hydroxyimine (imidic acid) followed by splitting of the dimer, and (iii) Z----E N-methylimine inversion. While pathway (iii----ii----i) is predicted as a feasible route terminating in the formation of a peptide bond, the inverse route (iii----ii----i) is excluded as a possible initial step in the generation of a 5(4H)-oxazolone intermediate.
J Comput Aided Mol Des 1991 Dec
PMID:Reaction mechanisms in peptide synthesis. Part 2. Tautomerism of the peptide bond. 181 93

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.
Mol Pharmacol 1991 Nov
PMID:Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C. 194 44

The effect of hyperammonemia of varying degree and duration on the gamma-glutamyl-transpeptidase (GGT) activity was studied in the homogenates and capillaries of different brain regions of the rat. "Acute" hyperammonemia (750 and 600 mg of ammonium acetate per kg b.w. were injected i.p. at 30 min interval, and the animals were decapitated immediately), in which blood ammonia was increased 14-fold, and brain ammonia six-fold above the control level, produced a 20% increase of the enzyme activity in cerebellum, and a 17% decrease in gyrus dentatus, but had no effect in the frontal cortex and the CA1 and CA3 regions of hippocampus. "Subchronic" hyperammonemia (two injections of 600 mg ammonium acetate/kg were given at 24 h intervals, and tissue samples were removed 24 h later), that was accompanied by only a 60% increase of blood or brain ammonia, increased the activity in cerebellum to 38% above control, but produced no effect in the other brain regions. "Chronic" hyperammonemia (three injections of 600 mg ammonium acetate/kg at 24 h intervals and excision of tissue samples 30 min after the last injection), in which blood and brain ammonia were, respectively, 60 and 100% higher than in control animals, elevated the GGT activity in the cerebellum by 57%, in CA1 by 15%, and in CA3 by 21%, but produced no effect in the frontal cortex or gyrus dentatus. By contrast, "chronic" hyperammonemia produced a 30% increase of GGT activity in cerebral cortical capillaries, but only a 10% increase in hippocampal capillaries, and no change in cerebellar capillaries. The results suggest that, hyperammonemia of relatively long duration may contribute to the enhancement of brain GGT activity observed in chronic forms of hepatic encephalopathy. However, ammonia does not appear to activate the enzyme directly.
Mol Chem Neuropathol
PMID:The effect of acute and repeated hyperammonemia on gamma-glutamyl transpeptidase in homogenates and capillaries of various rat brain regions. 198 79

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.
J Mol Biol 1991 Apr 05
PMID:Structure and evolution of a group of related aminoacyl-tRNA synthetases. 201 46

Many photosynthetic bacteria from aquatic and terrestrial habitats reduce atmospheric dinitrogen to ammonia. The synthesis of proteins required for nitrogen fixation in these microorganisms is repressed by fixed nitrogen or oxygen. Studies on the purple non-sulphur phototroph Rhodobacter capsulatus have helped to clarify this transcriptional control and to define the factors involved in this regulation. The molecular mechanisms by which the nitrogen and oxygen status of the cell are relayed into nif gene expression or repression involve many trans- and cis-acting factors. The roles of these factors in the nif regulatory cascade of R. capsulatus are summarized. Two levels of control are present. The first level of control involves the nitrogen sensing circuitry in which at least four proteins act in a cascade. Upon nitrogen deficiency, genes involved in the second level of control are transcriptionally activated. These genes encode regulatory proteins that subsequently activate transcription of all other nif genes under anaerobic conditions. The R. capsulatus cascade is compared to the nif regulatory cascade in Klebsiella pneumoniae, highlighting both common and unique aspects.
Mol Microbiol 1990 Nov
PMID:Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors. 208 42


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