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Query: UNIPROT:P06889 (Mol)
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The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.
J Mol Biol 1991 Mar 20
PMID:X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa. 190 63

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.
Mol Cell Biol 1991 Jun
PMID:Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis. 190 40

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.
Mol Cell Biol 1991 Jul
PMID:Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains. 190 42

We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.
Mol Cell Biol 1991 Jul
PMID:AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae. 190 46

We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, HO, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpd1 delta mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.
Mol Cell Biol 1991 Dec
PMID:RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes. 194 90

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.
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PMID:Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD. 197 79

Low-angle X-ray scattering in solution has been used to probe the quaternary structure of a mutant version of Escherichia coli aspartate transcarbamylase in which Glu239 of the catalytic chain was replaced by glutamine by site-directed mutagenesis. X-ray crystallographic studies of the wild-type enzyme have shown that one set of intersubunit interactions involving Glu239 are lost, and are replaced by another set of intrachain interactions when the enzyme undergoes the allosteric transition from the T to the R state. Functional analysis of the mutant enzyme with glutamine in place of Glu239 indicates that homotropic co-operativity is lost without altering the maximal specific activity. The radius of gyration of the unligated mutant enzyme is larger than the unligated wild-type, indicating an alteration in quaternary structure of the mutant. However, the radius of gyration of the mutant enzyme in the presence of N-(phosphonoacetyl)-L-aspartate (PALA) is identical with the value for the wild-type enzyme in the presence of PALA. X-ray scattering at larger angles indicates that the mutant enzyme is in a new structural state different from the wild-type T and R structures. The scattering pattern in the presence of saturating concentrations of PALA is identical with that of the wild-type R structure. Saturating concentrations of carbamyl phosphate alone are sufficient to convert most of the mutant enzyme to the R structure, in the absence of aspartate. CTP shifts the scattering pattern of the mutant enzyme in the presence of saturating carbamyl phosphate towards the scattering curve of the unligated enzyme, but CTP has no effect on the scattering curve in the absence of carbamyl phosphate or in the presence of subsaturating PALA. However, in the presence of subsaturating PALA, ATP causes a strong shift towards the R structure. Neither ATP nor CTP has any effect on the activity of the mutant enzyme. These data suggest that the replacement of Glu239 by glutamine results in a new quaternary structure. These data also explain, on a structural basis, why co-operativity is lost in this mutant enzyme.
J Mol Biol 1990 Jul 05
PMID:Structural consequences of the replacement of Glu239 by Gln in the catalytic chain of Escherichia coli aspartate transcarbamylase. 197 63

The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%-49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.
Mol Gen Genet 1990 Apr
PMID:Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: phylogenetic implications. 197 23

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.
Mol Cell Biol 1990 Dec
PMID:Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes. 197 25

Glutamate in glutamatergic neurons exists in a cytosolic pool, as well as a transmitter pool, which is assumed to be localized in synaptic vesicles. Transmitter glutamate released from glutamatergic neurons is taken up by both neurons and glial cells, giving rise to a flux of glutamate from neurons to astrocytes. In astrocytes, glutamine is formed from glutamate by the glial-specific enzyme glutamine synthetase (EC 6.3.1.2). Glutamine diffuses back to neurons, where glutamate is formed by phosphate-activated glutaminase (EC 3.5.1.2). However, this cycle is not stoichiometric, and glutamine obtained from glial cells cannot replenish all transmitter glutamate lost from neurons. 2-Oxoglutarate is another putative precursor for transmitter glutamate. Net synthesis of citric acid cycle intermediates is dependent on carbon dioxide fixation to pyruvate, catalyzed by pyruvate carboxylase (EC 6.4.1.1). Since this enzyme is exclusively glial, a net flow of citric acid cycle intermediates from glial cells to neurons probably exists. The quantitative contribution of each transmitter precursor may not be the same in different regions of the brain and may vary with the metabolic state of the neuron. The pool of transmitter glutamate is most likely regulated by the activity of glutamate-forming enzymes in the nerve terminal, and/or by uptake/release of glutamate and glutamate precursors through the synaptosomal plasma membrane.
Mol Chem Neuropathol 1990 Jan
PMID:Synthesis of transmitter glutamate and the glial-neuron interrelationship. 198 May 84


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