Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

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Laakso Kati

2007-10-01

Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

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Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

1976-07-01

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

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Hoff-Risseti, Caroline; Dörr, Felipe Augusto; Schaker, Patricia Dayane Carvalho; Pinto, Ernani; Werner, Vera Regina; Fiore, Marli Fatima

2013-01-01

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins. PMID:24015317

Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

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Caroline Hoff-Risseti

Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

1986-01-01

of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined......Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Taylor, G.R.; Barclay, B.J.; Storms, R.K.; Friesen, J.D.; Haynes, R.H.

1982-01-01

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp/sup +/ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy/sup +/ transformants directly, it was found that all pTL1 transformants were phenotypically Thy/sup +/ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-/sup 3/H]dUMP to [6-/sup 3/H]dTMP. In protein extracts from the thymidylate auxotroph (tmpl-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain

Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

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Parker, J; Fishman, S E

1979-04-01

The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA.

Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

Science.gov (United States)

Parker, J; Fishman, S E

1979-01-01

The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA. PMID:374370

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine.

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Ivanov, Ana; Kameka, Alexander; Pajak, Agnieszka; Bruneau, Luanne; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

2012-06-01

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

International Nuclear Information System (INIS)

Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

1983-01-01

A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

DEFF Research Database (Denmark)

Martinussen, Jan; Hammer, Karin

1998-01-01

The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown...

Preparation of sup(99m) Tc - L - asparagine

International Nuclear Information System (INIS)

Okada, H.; Silva, C.P.G. da; Pereira, N.P.S. de.

1986-05-01

The interrelationship between technetium-99m, different reducing agents and the L-asparagine is studied. The maximum labelling yield, aboult 95%, is obtained by using the following molar ratios for ligand to reducing agents: L-asparagine: stannous ascorbate, 150:1; the L-asparagine: ascorbic acid, 50:1, and L-asparagine:stannous chloride, 800:1. This radiopharmaceutical is used for visualization of dependent asparagine tumours. (Author) [pt

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

Energy Technology Data Exchange (ETDEWEB)

Bernard, S.M.; Habash, D.Z.

2009-07-02

Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

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Nederbragt Alexander J

2009-08-01

Full Text Available Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS. Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454" and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully

Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

1986-01-01

Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...... the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator....

A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

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Ashley L Waldron

Full Text Available Histidyl tRNA Synthetase (HARS is a member of the aminoacyl tRNA synthetase (ARS family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

Proteome reference maps of the Lotus japonicus nodule and root

DEFF Research Database (Denmark)

Dam, Svend Secher; Dyrlund, Thomas F.; Ussatjuk, Anna

2014-01-01

formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7...... stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous...

A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development.

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Olivier Silvie

2008-06-01

Full Text Available Plasmodium sporozoites invade host hepatocytes and develop as liver stages (LS before the onset of erythrocytic infection and malaria symptoms. LS are clinically silent, and constitute ideal targets for causal prophylactic drugs and vaccines. The molecular and cellular mechanisms underlying LS development remain poorly characterized. Here we describe a conserved Plasmodium asparagine-rich protein that is specifically expressed in sporozoites and liver stages. Gene disruption in Plasmodium berghei results in complete loss of sporozoite infectivity to rodents, due to early developmental arrest after invasion of hepatocytes. Mutant sporozoites productively invade host cells by forming a parasitophorous vacuole (PV, but subsequent remodelling of the membrane of the PV (PVM is impaired as a consequence of dramatic down-regulation of genes encoding PVM-resident proteins. These early arrested mutants confer only limited protective immunity in immunized animals. Our results demonstrate the role of an asparagine-rich protein as a key regulator of Plasmodium sporozoite gene expression and LS development, and suggest a requirement of partial LS maturation to induce optimal protective immune responses against malaria pre-erythrocytic stages. These findings have important implications for the development of genetically attenuated parasites as a vaccine approach.

Formation of 14C-asparagine from 14C-precursor in mulberry leaves

International Nuclear Information System (INIS)

Yamashita, Tadaaki

1981-01-01

Since a remarkable accumulation of asparagine in the young leaves of mulberry has been observed, the formation of 14 C-asparagine from 14 C-labeled substrates in young leaves was examined in comparison with that in the mature leaves. 14 C-aspartic acid and 14 C-succinic acid expected as active precursors for asparagine biosynthesis, and 14 C-sucrose as respiratory substrates were fed respectively to the disks of young or mature leaves of mulberry. Although 14 C-succinic acid was actively converted to 14 C-asparagine, no significant amount of 14 C-asparagine was formed from 14 C-aspartic acid in two hours of feeding period. The rate of formation of 14 C-asparagine from 14 C-succinic acid in the mature leaves was slightly higher than that in the young leaves. Amino acids other than asparagine acquired 14 C from 14 C-labeled substrates were mainly aspartic acid, glutamic acid, alanine and ν-amino butyric acid in both of the leaves. Intending to accelerate the formation of asparagine in the leaves, ammonium ion was supplied to culturing solution as only source of nitrogen and plants were grown for two weeks in that solution before 14 C-labeled substrates feeding experiments. Supplying of ammonium ion brought about enhanced accumulation of asparagine in the young leaves, and caused remarkable formation of 14 C-asparagine from 14 C-aspartic acid in both of the leaves. However, the rate of 14 C-asparagine formation from 14 C-aspartic acid in the young leaves did not exceed that in the mature leaves. (author)

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

Science.gov (United States)

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

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Seabra Ana R

2010-08-01

Full Text Available Abstract Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS, occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. Results This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2 in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. Conclusions This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

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Apfel, C M; Takács, B; Fountoulakis, M; Stieger, M; Keck, W

1999-01-01

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

Science.gov (United States)

Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

A highly conserved basidiomycete peptide synthetase produces a trimeric hydroxamate siderophore.

Science.gov (United States)

Brandenburger, Eileen; Gressler, Markus; Leonhardt, Robin; Lackner, Gerald; Habel, Andreas; Hertweck, Christian; Brock, Matthias; Hoffmeister, Dirk

2017-08-25

The model white-rot basidiomycete Ceriporiopsis ( Gelatoporia ) subvermispora B encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet uncharacterized fungal type VI siderophore synthetase family which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module and two thiolation/condensation (T-C) di-domain partial modules which, together, constitute an AT 1 C 1 T 2 C 2 T 3 C 3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as polyhistidine fusion in Aspergillus niger as soluble and active protein. N 5 -acetyl- N 5 -hydroxy-l-ornithine (l-AHO) and N 5 - cis -anhydromevalonyl- N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as substrates, as assessed in vitro using the substrate-dependent [ 32 P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo -CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as product in vitro , which was verified by LC-HRESIMS. Phylogenetic analyses suggest that type VI family siderophore synthetases are widespread in mushrooms and have evolved in a common ancestor of basidiomycetes. Importance : The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 supports further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the

Identification and Quantitation of Asparagine and Citrulline Using High-Performance Liquid Chromatography (HPLC)

OpenAIRE

Bai, Cheng; Reilly, Charles C.; Wood, Bruce W.

2007-01-01

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them...

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

Science.gov (United States)

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

Science.gov (United States)

... belongs to a class of genetic diseases called urea cycle disorders. In this condition, the carbamoyl phosphate synthetase I ... Management Resources (4 links) Baby's First Test GeneReview: Urea Cycle Disorders Overview MedlinePlus Encyclopedia: Hereditary Urea Cycle Abnormality National ...

Identification and quantitation of asparagine and citrulline using high-performance liquid chromatography (HPLC).

Science.gov (United States)

Bai, Cheng; Reilly, Charles C; Wood, Bruce W

2007-03-28

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (microMol ml(-1)/microMol ml(-1))], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.

Identification and Quantitation of Asparagine and Citrulline Using High-Performance Liquid Chromatography (HPLC

Directory of Open Access Journals (Sweden)

Cheng Bai

2007-01-01

Full Text Available High-performance liquid chromatography (HPLC analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates. Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol ml–1/μMol ml–1], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh. K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.

The standard enthalpies of formation of L-asparagine and L-{alpha}-glutamine

Energy Technology Data Exchange (ETDEWEB)

Contineanu, Iulia, E-mail: icontineanu@yahoo.com [Institute of Physical Chemistry ' I.G. Murgulescu' , Spl. Independentei 202, Bucharest (Romania); Neacsu, Ana, E-mail: anna_matache@yahoo.com [Institute of Physical Chemistry ' I.G. Murgulescu' , Spl. Independentei 202, Bucharest (Romania); Perisanu, Stefan T., E-mail: stefan.perisanu@upb.ro [Laboratory of General Chemistry, Polytechnic University of Bucharest, str. Polizu nr. 1, Bucuresti (Romania)

2010-01-10

The energies of combustion of L-asparagine and L-{alpha}-glutamine were measured in a static bomb adiabatic calorimeter. Corrections were made for the heats due to the ignition of sample and for the nitric acid formation. The derived enthalpies of formation in solid state of asparagine monohydrate, nonhydrated asparagine and glutamine are respectively -1084.1 {+-} 3.0, -788.1 {+-} 4.7 and -834.3 {+-} 4.6 kJ mol{sup -1}. The data of enthalpy of formation are compared with the literature values and with estimated values by means of group additivity, using parameters recommended by Domalski and Hearing. The discrepancies between experimental and calculated values are explained by considering the number and strength of intermolecular hydrogen bonds. The dehydration of asparagine monohydrate and the possible melting of the three amino acids were investigated by means of DSC. Glutamine melts without considerable decomposition at about 182 {sup o}C, while asparagines decompose during the fusion process (208 {sup o}C).

A transgenic approach to study argininosuccinate synthetase gene expression

Science.gov (United States)

2014-01-01

Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage

A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

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Paula Bustamante

2016-06-01

Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene

DEFF Research Database (Denmark)

hart, Leen M; Hansen, Torben; Rietveld, Ingrid

2005-01-01

Previously, we have shown that a mutation in the mitochondrial DNA-encoded tRNA(Leu(UUR)) gene is associated with type 2 diabetes. One of the consequences of this mutation is a reduced aminoacylation of tRNA(Leu(UUR)). In this study, we have examined whether variants in the leucyl tRNA synthetase...... gene (LARS2), involved in aminoacylation of tRNA(Leu(UUR)), associate with type 2 diabetes. Direct sequencing of LARS2 cDNA from 25 type 2 diabetic subjects revealed eight single nucleotide polymorphisms. Two of the variants were examined in 7,836 subjects from four independent populations...... in the Netherlands and Denmark. A -109 g/a variant was not associated with type 2 diabetes. Allele frequencies for the other variant, H324Q, were 3.5% in type 2 diabetic and 2.7% in control subjects, respectively. The common odds ratio across all four studies was 1.40 (95% CI 1.12-1.76), P = 0.004. There were...

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

Energy Technology Data Exchange (ETDEWEB)

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

Science.gov (United States)

Prinsi, Bhakti; Espen, Luca

2015-04-03

Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post

Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

DEFF Research Database (Denmark)

Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

1995-01-01

The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...

Model studies on acrylamide generation from glucose/asparagine in aqueous glycerol

DEFF Research Database (Denmark)

Hedegaard, Rikke Susanne Vingborg; Frandsen, Henrik Lauritz; Granby, Kit

2007-01-01

Acrylamide formation from asparagine and glucose in different ratios in neutral glycerol/water mixtures was found to increase with decreasing water activity (0.33......Acrylamide formation from asparagine and glucose in different ratios in neutral glycerol/water mixtures was found to increase with decreasing water activity (0.33...

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

Science.gov (United States)

Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

2016-08-01

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Cerebrospinal fluid asparagine depletion during pegylated asparaginase therapy in children with acute lymphoblastic leukaemia

DEFF Research Database (Denmark)

Henriksen, Louise Tram; Nersting, Jacob; Raja, Raheel A

2014-01-01

L-asparaginase is an important drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Cerebrospinal fluid (CSF) asparagine depletion is considered a marker of asparaginase effect in the central nervous system (CNS) and may play a role in CNS-directed anti-leukaemia therapy. The o...... in CSF asparagine corresponded to serum enzyme activities above 50 iu/l. Higher serum enzyme activities were not followed by more extensive depletion. In conclusion, pegylated asparaginase 1000 iu/m(2) i.m. every second week effectively reduced CSF asparagine levels.......L-asparaginase is an important drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Cerebrospinal fluid (CSF) asparagine depletion is considered a marker of asparaginase effect in the central nervous system (CNS) and may play a role in CNS-directed anti-leukaemia therapy....... The objective of this study was to describe CSF asparagine depletion during 30 weeks of pegylated asparaginase therapy, 1000 iu/m(2) i.m. every second week, and to correlate CSF asparagine concentration with serum L-asparaginase enzyme activity. Danish children (1-17 years) with ALL, treated according...

Role of curcumin in the conversion of asparagine into acrylamide during heating.

Science.gov (United States)

Hamzalıoğlu, Aytül; Mogol, Burçe A; Lumaga, Roberta Barone; Fogliano, Vincenzo; Gökmen, Vural

2013-06-01

This study aimed to investigate the ability of curcumin to convert asparagine into acrylamide during heating at different temperatures. Binary and ternary model systems of asparagine-curcumin and asparagine-curcumin-fructose were used to determine the role of curcumin on acrylamide formation in competitive and uncompetitive reaction conditions. The results indicated that curcumin could potentially contribute to acrylamide formation under long-term heating conditions as long as asparagine was present in the medium. The amount of acrylamide formed in the ternary system was slightly higher than in the binary system during heating (p available. The kinetic trends were similar in both model systems evidencing that fructose reacted with asparagine more rapidly than curcumin. The data reveal that acrylamide formation in the temperature range of 150-200°C obeys Arrhenius law with activation energy of 79.1 kJ/mole. Data of this work showed the possibility that antioxidants having a carbonyl compound can react directly with ASN leading to acrylamide. The addition of antioxidants to foods may increase the formation of acrylamide upon long-term heating if free sugar concentration is low and ASN concentration is relatively high.

Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

International Nuclear Information System (INIS)

Pendergast, A.M.

1986-01-01

The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

Science.gov (United States)

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

Science.gov (United States)

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus.

Science.gov (United States)

Reeves, Emer P; Reiber, Kathrin; Neville, Claire; Scheibner, Olaf; Kavanagh, Kevin; Doyle, Sean

2006-07-01

Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF/TOF MS (four internal peptides sequenced). A pes1 disruption mutant (delta pes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The delta pes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P = 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.

Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

DEFF Research Database (Denmark)

O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

2012-01-01

The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end produc...

ATF5 polymorphisms influence ATF function and response to treatment in children with childhood acute lymphoblastic leukemia

OpenAIRE

Rousseau, Julie; Gagné, Vincent; Labuda, Malgorzata; Beaubois, Cyrielle; Sinnett, Daniel; Laverdière, Caroline; Moghrabi, Albert; Sallan, Stephen E.; Silverman, Lewis B.; Neuberg, Donna; Kutok, Jeffery L.; Krajinovic, Maja

2011-01-01

Asparaginase is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia. Asparagine synthetase (ASNS) and the basic region leucine zipper activating transcription factor 5 (ATF5) and arginosuccinate synthase 1 (ASS1) have been shown to mediate the antileukemic effect of asparaginase and to display variable expression between leukemia cells that are resistant and sensitive to treatment. Fourteen polymorphisms in the regulatory and coding regions of these gene...

Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

DEFF Research Database (Denmark)

Bernard, Stéphanie M.; Møller, Anders Laurell Blom; Dionisio, Giuseppe

2008-01-01

). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular......We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...... sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle...

Toward a Kinetic Model for Acrylamide Formation in a Glucose-Asparagine Reaction System

NARCIS (Netherlands)

Knol, J.J.; Loon, W.A.M.; Linssen, J.P.H.; Ruck, A.L.; Boekel, van M.A.J.S.

2005-01-01

A kinetic model for the formation of acrylamide in a glucose-asparagine reaction system is pro-posed. Equimolar solutions (0.2 M) of glucose and asparagine were heated at different tempera-tures (120-200 C) at pH 6.8. Besides the reactants, acrylamide, fructose, and melanoidins were quantified after

Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

Directory of Open Access Journals (Sweden)

Ye Eun Kim

2016-11-01

Full Text Available It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes. The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates, and non-functional mutants of the two sequences (which act as parasites. Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

Construction of hybrid peptide synthetases by module and domain fusions.

Science.gov (United States)

Mootz, H D; Schwarzer, D; Marahiel, M A

2000-05-23

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

Preparation of L-asparagine complex with technetium

International Nuclear Information System (INIS)

Persano, S.C.M.

1981-01-01

The preparation of technetium chelated to L-asparagine is aimed at for obtention of potentially useful radiopharmaceutical in nuclear medicine. The reduction of pertechnetate anion ( 99 TcO - 4 and sup(99m) TcO - 4 ) using hydrazine as a reducing agent is studied with use of polarographic spectrophotometric and chromatographic methods. Spectrophotometric determination shows that the aqueous solution of amonium sup(-99m) Tc pertechnetate absorbs at 244, 248 and 290 nm. After reduction the species absorbs at 330 and near 495-530 nm. Polarographic measurements with 0,1 N NaOH electrolyte show a half-wave potential E sub(1/2 = -0,82 V for technetium +VII. After the addition of increasing amounts of hydrazine into a solution containing Tc+VII, the half wave potential shifts to more positives values, indicating the reduction of Tc+VII. Chromatographic determinations are in good agreement with polarographic analysis, emphatizing reduction of Tc+IV by hydrazine. The reduced 99 Tc shows ability of being incorporated into L-asparagine forming a cristalline compound with melting point 118.6 0 C. Infrared absorption spectrometry shows that amino and carboxyl groups are bound to the metal in this complex. The yield of sup(99m) Tc incorporation into L-asparagine at 60 0 C in 30 minutes is up to 95%. The labeled complex can be presented as a radiopharmaceutical product. Tissue distribution performed in groups of normal and bearing lymphoma Walker 256 Wistar rats (50-60) shows that the radiopharmaceutical concentrates seletively in the tumor and is fastly excreted by the kidney and it doesn't have significant affinity for any organ, being so adequate for the formation of tumoral images. (Authors) [pt

Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

DEFF Research Database (Denmark)

Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

1988-01-01

in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional...

Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen-use- and yield-related traits in bread wheat.

Science.gov (United States)

Li, Xin-Peng; Zhao, Xue-Qiang; He, Xue; Zhao, Guang-Yao; Li, Bin; Liu, Dong-Cheng; Zhang, Ai-Min; Zhang, Xue-Yong; Tong, Yi-Ping; Li, Zhen-Sheng

2011-01-01

Glutamine synthetase (GS) plays a key role in the growth, nitrogen (N) use and yield potential of cereal crops. Investigating the haplotype variation of GS genes and its association with agronomic traits may provide useful information for improving wheat N-use efficiency and yield. We isolated the promoter and coding region sequences of the plastic glutamine synthetase isoform (GS2) genes located on chromosomes 2A, 2B and 2D in bread wheat. By analyzing nucleotide sequence variations of the coding region, two, six and two haplotypes were distinguished for TaGS2-A1 (a and b), TaGS2-B1 (a-f) and TaGS2-D1 (a and b), respectively. By analyzing the frequency data of different haplotypes and their association with N use and agronomic traits, four major and favorable TaGS2 haplotypes (A1b, B1a, B1b, D1a) were revealed. These favorable haplotypes may confer better seedling growth, better agronomic performance, and improved N uptake during vegetative growth or grain N concentration. Our data suggest that certain TaGS2 haplotypes may be valuable in breeding wheat varieties with improved agronomic performance and N-use efficiency. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Science.gov (United States)

Souza, Sarah C R; Mazzafera, Paulo; Sodek, Ladaslav

2016-05-01

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2 defects predicts differential effects on aminoacylation

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Liliya eEuro

2015-02-01

Full Text Available The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19 is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations.The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change p.R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

Science.gov (United States)

Sun, Wei-Wen; Guo, Chun-Jun; Wang, Clay C C

2016-04-01

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

Science.gov (United States)

Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

The influence of fermented deffated flaxseed with difference lactic acid bacteria's on asparagine and acrylamide content in biscuits.

OpenAIRE

Bukauskienė, Gintarė

2016-01-01

It was evaluated the influence of solid state (SSF) and traditional (TF) fermentation with different lactobacillus (LAB) defatted flaxseeds on asparagine and acrylamide content in biscuits, and analysed correlation between fermented supplements physico chemical indicators and biscuits color and their sensory properties on acrylamide and asparagine content in biscuits. Correliations betweem acrylamide and asparagine content in biscuits and fermented supplements acidity parameters (pH, total t...

Fusion of the subunits α and β of succinyl-CoA synthetase as a phylogenetic marker for Pezizomycotina fungi

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Amanda M. Koire

2011-01-01

Full Text Available Gene fusions, yielding the formation of multidomain proteins, are evolutionary events that can be utilized as phylogenetic markers. Here we describe a fusion gene comprising the α and β subunits of succinyl-coA synthetase, an enzyme of the TCA cycle, in Pezizomycotina fungi. This fusion is present in all Pezizomycotina with complete genome sequences and absent from all other organisms. Phylogenetic analysis of the α and β subunits of succinyl-CoA synthetase suggests that both subunits were duplicated and retained in Pezizomycotina while one copy was lost from other fungi. One of the duplicated copies was then fused in Pezizomycotina. Our results suggest that the fusion of the α and β subunits of succinyl-CoA synthetase can be used as a molecular marker for membership in the Pezizomycotina subphylum. If a species has the fusion it can be reliably classified as Pezizomycotina, while the absence of the fusion is suggestive that the species is not a member of this subphylum.

Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

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Nussinov Ruth

2006-03-01

Full Text Available Abstract Background While the premise that lateral gene transfer (LGT is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS is found both as a class 1 and a class 2 enzyme (LysRS1-2. Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention. Results We applied a phylogenetic approach for determining the extent and origin of LGT in prokaryotic LysRS. Reconstructing species trees for Archaea and Bacteria, and inferring that their last common ancestors encoded LysRS1 and LysRS2, respectively, we studied the gains and losses of both classes. A complex pattern of LGT events emerged. In specific groups of organisms LysRS1 was replaced by LysRS2 (and vice versa. In one occasion, within the alpha proteobacteria, a LysRS2 to LysRS1 LGT was followed by reversal to LysRS2. After establishing the most likely LGT paths, we studied the possible origins of the laterally transferred genes. To this end, we reconstructed LysRS gene trees and evaluated the likely origins of the laterally transferred genes. While the sources of LysRS1 LGTs were readily identified, those for LysRS2 remain, for now, uncertain. The replacement of one LysRS by another apparently transits through a stage simultaneously coding for both synthetases, probably conferring a selective advantage to the affected organisms. Conclusion The family of LysRSs features complex LGT events. The currently available data were sufficient for identifying unambiguously the origins of LysRS1 but not of LysRS2 gene transfers. A selective advantage is suggested to organisms encoding simultaneously LysRS1-2.

Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition.

Science.gov (United States)

Wang, Y H; Garvin, D F; Kochian, L V

2001-09-01

A subtractive tomato (Lycopersicon esculentum) root cDNA library enriched in genes up-regulated by changes in plant mineral status was screened with labeled mRNA from roots of both nitrate-induced and mineral nutrient-deficient (-nitrogen [N], -phosphorus, -potassium [K], -sulfur, -magnesium, -calcium, -iron, -zinc, and -copper) tomato plants. A subset of cDNAs was selected from this library based on mineral nutrient-related changes in expression. Additional cDNAs were selected from a second mineral-deficient tomato root library based on sequence homology to known genes. These selection processes yielded a set of 1,280 mineral nutrition-related cDNAs that were arrayed on nylon membranes for further analysis. These high-density arrays were hybridized with mRNA from tomato plants exposed to nitrate at different time points after N was withheld for 48 h, for plants that were grown on nitrate/ammonium for 5 weeks prior to the withholding of N. One hundred-fifteen genes were found to be up-regulated by nitrate resupply. Among these genes were several previously identified as nitrate responsive, including nitrate transporters, nitrate and nitrite reductase, and metabolic enzymes such as transaldolase, transketolase, malate dehydrogenase, asparagine synthetase, and histidine decarboxylase. We also identified 14 novel nitrate-inducible genes, including: (a) water channels, (b) root phosphate and K(+) transporters, (c) genes potentially involved in transcriptional regulation, (d) stress response genes, and (e) ribosomal protein genes. In addition, both families of nitrate transporters were also found to be inducible by phosphate, K, and iron deficiencies. The identification of these novel nitrate-inducible genes is providing avenues of research that will yield new insights into the molecular basis of plant N nutrition, as well as possible networking between the regulation of N, phosphorus, and K nutrition.

New perspectives on glutamine synthetase in grasses.

Science.gov (United States)

Swarbreck, Stéphanie M; Defoin-Platel, M; Hindle, M; Saqi, M; Habash, Dimah Z

2011-02-01

Members of the glutamine synthetase (GS) gene family have now been characterized in many crop species such as wheat, rice, and maize. Studies have shown that cytosolic GS isoforms are involved in nitrogen remobilization during leaf senescence and emphasized a role in seed production particularly in small grain crop species. Data from the sequencing of genomes for model crops and expressed sequence tag (EST) libraries from non-model species have strengthened the idea that the cytosolic GS genes are organized in three functionally and phylogenetically conserved subfamilies. Using a bioinformatic approach, the considerable publicly available information on high throughput gene expression was mined to search for genes having patterns of expression similar to GS. Interesting new hypotheses have emerged from searching for co-expressed genes across multiple unfiltered experimental data sets in rice. This approach should inform new experimental designs and studies to explore the regulation of the GS gene family further. It is expected that understanding the regulation of GS under varied climatic conditions will emerge as an important new area considering the results from recent studies that have shown nitrogen assimilation to be critical to plant acclimation to high CO(2) concentrations.

Reaction Intermediate Analogues as Bisubstrate Inhibitors of Pantothenate Synthetase

OpenAIRE

Xu, Zhixiang; Yin, Wei; Martinelli, Leonardo K.; Evans, Joanna; Chen, Jinglei; Yu, Yang; Wilson, Daniel J.; Mizrahi, Valerie; Qiao, Chunhua; Aldrich, Courtney C.

2014-01-01

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β–alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the in...

Physical studies of adenylosuccinate synthetase

International Nuclear Information System (INIS)

Bass, M.B.

1987-01-01

To determine the chemical mechanism of the reaction catalyzed by adenylosuccinate synthetase, positional isotope exchange studies were performed. Positional isotope exchange from the β-γ bridge to the β nonbridge position of [γ- 18 O]GTP was followed using 31 P NMR. The positional isotope exchange was found to occur in the presence of either IMP or IMP and succinate. The exchange did not occur in the presence of asparate. These results support a reaction mechanism which involves formation of a 6-phosphoryl-IMP intermediate with subsequent attack by aspartate to form adenylosuccinate as originally proposed by Lieberman in 1956. In order to resolve the NMR resonances for positional isotope exchange, it was necessary to find a chelator which would limit exchange broadening. trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid was found to be a suitable chelator at neutral and acidic pH. Studies of adenylosuccinate synthetase from Escherichia coli have been limited by the low concentrations of enzyme present in the cell and the difficulty in purifying the enzyme to homogeneity. Overproduction of the enzyme by cloning the purA gene into a runaway replication plasmid allowed the cells to produce a much higher concentration of enzyme. A new purification scheme is reported that takes advantage of the overproduced enzyme. Yields of 75 mg of homogeneous enzyme have been obtained from 76 g of E. coli cell paste

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

OpenAIRE

Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

1998-01-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and...

The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a.

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Juan Jesús Molina-Rueda

Full Text Available BACKGROUND: Glutamine synthetase (GS plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4 expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. RESULTS: In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs. The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs, with concomitant up-regulation of plastidic iron SODs (FSDs in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated down-regulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. CONCLUSIONS: Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

Science.gov (United States)

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere.

Science.gov (United States)

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Carotenogenesis and asparagine in leptosphaeria michotii (West) sacc

International Nuclear Information System (INIS)

Jerebzoff-Quintin, S.; Jerebzoff, St.

1980-01-01

In Leptosphaeria michotii U 14 C-asparagine was incorporated into the coloured carotenoids, the synthesis of which carried on till day 8. The pigment turnover, obvious from day 6, was not modified by the light conditions used. Nicotine (0.25 to 4.5mM) has been used to study carotenogenesis and sporulation rhythm regulation in L. michotii fed with asparagine 2.6mM control cultures contained in darkness β-carotene only and in continuous light β-carotene 98% and lycopene 2%. The mold receiving nicotine 0.25mM in darkness contained β-carotene 98% and lycopene 2%. For nicotine 0.5mM and upwards β-carotene decreased, lycopene increased and γ-carotene appeared, the balance between these pigments also depending on the light conditions. Whereas period length (tau) of the sporulation rhythm increased from one cycle to the next in control cultures in darkness, it was stabilized either by continuous light (tau27h) or by nicotine 0.25mM (tau30h). For nicotine >= 0.5mM sporulation was uniform in darkness or in light. (author)

Distribution of events of positive selection and population differentiation in a metabolic pathway: the case of asparagine N-glycosylation

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Dall’Olio Giovanni

2012-06-01

Full Text Available Abstract Background Asparagine N-Glycosylation is one of the most important forms of protein post-translational modification in eukaryotes. This metabolic pathway can be subdivided into two parts: an upstream sub-pathway required for achieving proper folding for most of the proteins synthesized in the secretory pathway, and a downstream sub-pathway required to give variability to trans-membrane proteins, and involved in adaptation to the environment and innate immunity. Here we analyze the nucleotide variability of the genes of this pathway in human populations, identifying which genes show greater population differentiation and which genes show signatures of recent positive selection. We also compare how these signals are distributed between the upstream and the downstream parts of the pathway, with the aim of exploring how forces of population differentiation and positive selection vary among genes involved in the same metabolic pathway but subject to different functional constraints. Results Our results show that genes in the downstream part of the pathway are more likely to show a signature of population differentiation, while events of positive selection are equally distributed among the two parts of the pathway. Moreover, events of positive selection are frequent on genes that are known to be at bifurcation points, and that are identified as being in key position by a network-level analysis such as MGAT3 and GCS1. Conclusions These findings indicate that the upstream part of the Asparagine N-Glycosylation pathway has lower diversity among populations, while the downstream part is freer to tolerate diversity among populations. Moreover, the distribution of signatures of population differentiation and positive selection can change between parts of a pathway, especially between parts that are exposed to different functional constraints. Our results support the hypothesis that genes involved in constitutive processes can be expected to show

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc.

Science.gov (United States)

Genuário, Diego Bonaldo; Silva-Stenico, Maria Estela; Welker, Martin; Beraldo Moraes, Luiz Alberto; Fiore, Marli Fátima

2010-04-01

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, São Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 16S rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 16S rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin. Copyright 2009 Elsevier Ltd. All rights reserved.

The expression of selected non-ribosomal peptide synthetases in Aspergillus fumigatus is controlled by the availability of free iron.

Science.gov (United States)

Reiber, Kathrin; Reeves, Emer P; Neville, Claire M; Winkler, Robert; Gebhardt, Peter; Kavanagh, Kevin; Doyle, Sean

2005-07-01

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SidE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD ( approximately 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy.

Kinetics of formation of acrylamide and Schiff base intermediates from asparagine and glucose

DEFF Research Database (Denmark)

Hedegaard, Rikke Susanne Vingborg; Frandsen, Henrik; Skibsted, Leif H.

2008-01-01

From the concentration of glucose and asparagine as reactants and of acrylamide as product each determined by LC-MS during reaction in an acetonitrile/water (68:32) model system at pH 7.6 (0.04 M phosphate buffer) and from the relative concentration of the Schiff base intermediate, the decarboxyl......From the concentration of glucose and asparagine as reactants and of acrylamide as product each determined by LC-MS during reaction in an acetonitrile/water (68:32) model system at pH 7.6 (0.04 M phosphate buffer) and from the relative concentration of the Schiff base intermediate...

Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

Science.gov (United States)

Sharma, Arvind; Sharma, Amit

2015-02-01

The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

Acrylamide and 5-hydroxymethylfurfural formation during biscuit baking. Part II: Effect of the ratio of reducing sugars and asparagine.

Science.gov (United States)

Nguyen, Ha T; van der Fels-Klerx, H J Ine; van Boekel, M A J S

2017-09-01

This study investigated acrylamide and 5-hydroxymethylfurfural (HMF) formation during biscuit baking. Four types of wheat flour with different molar ratios of total fructose and glucose to asparagine were investigated. Nevertheless, the molar ratio in all four biscuit doughs exceeded one after proofing due to enzyme action. Data obtained after baking were used to develop a mechanistic model, based on the asparagine-related pathway, for acrylamide and HMF formation in the four baked biscuit types. Asparagine reacted with fructose to form a Schiff base before decarboxylation to produce acrylamide without Amadori rearrangement product and sugar fragmentation. Fructose contributed considerably to acrylamide formation and to HMF formation via caramelization in all four biscuit types. No clear correlation was found between acrylamide and HMF in baked biscuits, nor between asparagine and the sum of glucose and fructose concentrations in the wheat flour. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

International Nuclear Information System (INIS)

Bandaralage, Sahan P.S.; Farnaghi, Soheil; Dulhunty, Joel M.; Kothari, Alka

2016-01-01

Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

Energy Technology Data Exchange (ETDEWEB)

Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

2016-03-15

Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro

International Nuclear Information System (INIS)

Woehle, D.L.; Lueddecke, B.A.; Ludden, P.W.

1990-01-01

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [α- 32 P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [α- 32 P]ATP- and [α- 32 P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [α- 32 P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes. A 32 P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H 3 32 PO 4 -grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification

Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

Science.gov (United States)

Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

2016-08-10

To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (PMSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. Copyright © 2016 Elsevier B.V. All rights reserved.

Ischemic preconditioning inhibits over-expression of arginyl-tRNA synthetase gene Rars in ischemia-injured neurons.

Science.gov (United States)

Shen, Yin; Zhao, Hong-Yang; Wang, Hai-Jun; Wang, Wen-Liang; Zhang, Li-Zhi; Fu, Rong

2016-08-01

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

Genes required for Lactococcus garvieae survival in a fish host.

Science.gov (United States)

Menéndez, Aurora; Fernández, Lucia; Reimundo, Pilar; Guijarro, José A

2007-10-01

Lactococcus garvieae is considered an emergent pathogen in aquaculture and it is also associated with mastitis in domestic animals as well as human endocarditis and septicaemia. In spite of this, the pathogenic mechanisms of this bacterium are poorly understood. Signature-tagged mutagenesis was used to identify virulence factors and to establish the basis of pathogen-host interactions. A library of 1250 L. garvieae UNIUD074-tagged Tn917 mutants in 25 pools was screened for the ability to grow in fish. Among them, 29 mutants (approx. 2.4 %) were identified which could not be recovered from rainbow trout following infection. Sequence analysis of the tagged Tn917-interrupted genes in these mutants indicated the participation in pathogenesis of the transcriptional regulatory proteins homologous to GidA and MerR; the metabolic enzymes asparagine synthetase A and alpha-acetolactate synthase; the ABC transport system of glutamine and a calcium-transporting ATPase; the dltA locus involved in alanylation of teichoic acids; and hypothetical proteins containing EAL and Eis domains, among others. Competence index experiments in several of the selected mutants confirmed the relevance of the Tn917-interrupted genes in the development of the infection process. The results suggested some of the metabolic routes and enzymic systems necessary for the complete virulence of this bacterium. This work is believed to represent the first report of a genome-wide scan for virulence factors in L. garvieae. The identified genes will further our understanding of the pathogenesis of L. garvieae infections and may provide targets for intervention or lead to the development of novel therapies.

Cytosolic glutamine synthetase in barley

DEFF Research Database (Denmark)

Thomsen, Hanne Cecilie

remobilisation from ageing plant parts. Thus, GS is highly involved in determining crop yield and NUE. The major objective of this PhD project was to investigate the NUE properties of transgenic barley designed to constitutively overexpress a GS1 isogene (HvGS1.1). These transgenic lines exhibited an increased...... for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root...... fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Science.gov (United States)

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

2014-01-01

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

Science.gov (United States)

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

2014-11-25

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

DEFF Research Database (Denmark)

Durkin, M E; Jäger, A C; Khurana, T S

1999-01-01

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome...... 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we...... found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein...

The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

Science.gov (United States)

Bieganowski, Pawel; Brenner, Charles

2003-08-29

Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

Science.gov (United States)

Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

2016-12-01

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response Constitutes a Pathogenic Strategy of group A Streptococcus

Directory of Open Access Journals (Sweden)

Emanuel eHanski

2014-08-01

Full Text Available The connection between bacterial pathogens and unfolded protein response (UPR is poorly explored. In this review we highlight the evidence showing that group A streptococcus (GAS induces endoplasmic reticulum (ER stress and UPR through which it captures the amino acid asparagine (ASN from the host. GAS acts extracellularly and during adherence to host cells it delivers the hemolysin toxins; streptolysin O (SLO and streptolysin S (SLS. By poorly understood pathways, these toxins trigger UPR leading to the induction of the transcriptional regulator ATF4 and consequently to the upregulation of asparagine synthetase (ASNS transcription leading to production and release of ASN. GAS senses ASN and alters gene expression profile accordingly, and increases the rate of multiplication. We suggest that induction of UPR by GAS and by other bacterial pathogens represent means through which bacterial pathogens gain nutrients from the host, obviating the need to become internalized or inflict irreversible cell damage.

Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects

NARCIS (Netherlands)

Santer, R.; Muhle, H.; Suormala, T.; Baumgartner, E. R.; Duran, M.; Yang, X.; Aoki, Y.; Suzuki, Y.; Stephani, U.

2003-01-01

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the

Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

Science.gov (United States)

León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

2017-08-21

The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

International Nuclear Information System (INIS)

Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro

1984-01-01

The antibiotic thiolactomycin inhibits the fatty acid synthesis from both [1- 14 C]-acetate and [2 14 C] malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases. (author)

Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

Energy Technology Data Exchange (ETDEWEB)

Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro (Tokyo Univ. (Japan). Faculty of Science)

1984-03-01

The antibiotic thiolactomycin inhibits the fatty acid synthesis from both (1-/sup 14/C)-acetate and (2/sup 14/C) malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases.

Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

Science.gov (United States)

Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

2013-01-01

Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

Science.gov (United States)

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

2008-11-25

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy

NARCIS (Netherlands)

Kempkes, L.J.M.; Martens, J.; Grzetic, J.; Berden, G.; Oomens, J.

2016-01-01

Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the

Mutations of the aminoacyl-tRNA-synthetases SARS and WARS2 are implicated in the etiology of autosomal recessive intellectual disability.

Science.gov (United States)

Musante, Luciana; Püttmann, Lucia; Kahrizi, Kimia; Garshasbi, Masoud; Hu, Hao; Stehr, Henning; Lipkowitz, Bettina; Otto, Sabine; Jensen, Lars R; Tzschach, Andreas; Jamali, Payman; Wienker, Thomas; Najmabadi, Hossein; Ropers, Hans Hilger; Kuss, Andreas W

2017-06-01

Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here, we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNA Ser concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the etiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions. © 2017 Wiley Periodicals, Inc.

Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI.

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Petersen, Lauren M; LaCourse, Kaitlyn; Schöner, Tim A; Bode, Helge; Tisa, Louis S

2017-11-01

Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA , which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli , swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA , these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

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Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

DEFF Research Database (Denmark)

Bonnevie-Nielsen, Vagn; Field, L Leigh; Lu, Shao

2005-01-01

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine......=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism...... at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA...

Asparagine and glycine metabolism in rat liver mitochondria and in mouse L5178Y lymphoma cells resistant or sensitive to the anticancer drug L-asparaginase

Energy Technology Data Exchange (ETDEWEB)

Keefer, J.F. Jr.

1986-01-01

Rat liver mitochondrial asparagine was found to be degraded via an aminotransferase and omega-amidase. Evidence includes oxaloacetate production from asparagine only when glyoxylate was added and production of radiolabeled ..cap alpha..-ketosuccinamate via metabolism of (U-/sup 14/C)asparagine. In the cytosol, asparagine is degraded primarily via asparaginase and subsequent transamination. A new HPLC technique for separation of citric acid cycle intermediates was developed using: ion pairing with 20 mM each to tetrabutylammonium hydroxide and Na/sub 2/SO/sub 4/; pH 7.0; isocratic elution; and detection at 210 nm. Amino acid content of mouse lymphoma cells either sensitive (L5178Y) or resistant (L5178Y/L-ASE) to the anticancer drug L-asparaginase was studied. The concentration of asparagine was 1.5 times higher and the concentrations of the essential amino acids histidine, methionine, valine and phenylalanine were two times higher in asparaginase-resistant than sensitive cells. In vivo but not in vitro studies indicated that glucine decreases in sensitive but not resistant cells upon asparaginase treatment. Asparagine and glycine metabolism was further studied using /sup 14/C radiolabel conversion of asparagine, glyoxylate, glycine and serine. Glycine metabolism is especially important in lymphomas and leukemias because these cells contain higher concentrations of glycine that other cancer and normal cells. Therefore, glycine levels were studied and were found to decrease in sensitive but not resistant cells upon asparaginase administration.

Potentiometric and spectral studies of complex formation of La(3), Pr(3) and Lu(3) with aspartic acid and asparagine

International Nuclear Information System (INIS)

Wojciechowska, A.; Lomozik, L.; Zielinski, S.

1987-01-01

The composition and stability of La 3+ , Pr 3+ and Lu 3+ complexes with aspartic acid and asparagine were analysed. The formation of complexes of the type ML and MHL was determined for La 3+ and Pr 3+ with aspartic acid, and of the type MHL for Lu 3+ with aspartic acid. For La 3+ , Pr 3+ and Lu 3+ with asparagine the formation of ML(OH) complexes was observed. By means of 1 HNMR and 13 CNMR studies the participation in the coordination of both -COOH groups was determined for aspartic acid, whereas for asparagine the participation of the -COOH group was determined in complexes with La 3+ , Pr 3+ , and of the -COOH and the -NH 2 groups in the complex with Lu 3+ . (Author)

Importance of asparagine on the conformational stability and chemical reactivity of selected anti-inflammatory peptides

Energy Technology Data Exchange (ETDEWEB)

Soriano-Correa, Catalina, E-mail: csorico@comunidad.unam.mx [Química Computacional, Facultad de Estudios Superiores (FES)-Zaragoza, Universidad Nacional Autónoma de México (UNAM), Iztapalapa, C.P. 09230 México, D.F. (Mexico); Barrientos-Salcedo, Carolina [Laboratorio de Química Médica y Quimiogenómica, Facultad de Bioanálisis Campus Veracruz-Boca del Río, Universidad Veracruzana, C.P. 91700 Veracruz (Mexico); Campos-Fernández, Linda; Alvarado-Salazar, Andres [Química Computacional, Facultad de Estudios Superiores (FES)-Zaragoza, Universidad Nacional Autónoma de México (UNAM), Iztapalapa, C.P. 09230 México, D.F. (Mexico); Esquivel, Rodolfo O. [Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa (UAM-Iztapalapa), C.P. 09340 México, D.F. (Mexico)

2015-08-18

Highlights: • Asparagine plays an important role to anti-inflammatory effect of peptides. • The electron-donor substituent groups favor the formation of the hydrogen bonds, which contribute in the structural stability of peptides. • Chemical reactivity and the physicochemical features are crucial in the biological functions of peptides. - Abstract: Inflammatory response events are initiated by a complex series of molecular reactions that generate chemical intermediaries. The structure and properties of peptides and proteins are determined by the charge distribution of their side chains, which play an essential role in its electronic structure and physicochemical properties, hence on its biological functionality. The aim of this study was to analyze the effect of changing one central amino acid, such as substituting asparagine for aspartic acid, from Cys–Asn–Ser in aqueous solution, by assessing the conformational stability, physicochemical properties, chemical reactivity and their relationship with anti-inflammatory activity; employing quantum-chemical descriptors at the M06-2X/6-311+G(d,p) level. Our results suggest that asparagine plays a more critical role than aspartic acid in the structural stability, physicochemical features, and chemical reactivity of these tripeptides. Substituent groups in the side chain cause significant changes on the conformational stability and chemical reactivity, and consequently on their anti-inflammatory activity.

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

Science.gov (United States)

Chadha, Sanya; Mallampudi, N Arjunreddy; Mohapatra, Debendra K; Madhubala, Rentala

2017-01-01

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase ( Ld LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. Ld LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized Ld LysRS-1. Recombinant Ld LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The Ld LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. Ld LysRS-1 appears to be an essential gene, as a chromosomal knockout of Ld LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC 50 ], 4.19 µM) and intracellular amastigotes (IC 50 , 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using Ld LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant Ld LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

Science.gov (United States)

Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

2015-08-01

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

Science.gov (United States)

Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

2016-02-13

Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

Energy Technology Data Exchange (ETDEWEB)

Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

2013-02-15

Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

Science.gov (United States)

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

Directory of Open Access Journals (Sweden)

Alexandre Gouzy

2014-02-01

Full Text Available Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s, that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

Science.gov (United States)

Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

2016-11-10

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

Science.gov (United States)

Aboelnga, Mohamed M.

Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre?transfer editing and post?transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyl-tRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

Science.gov (United States)

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

Directory of Open Access Journals (Sweden)

A Theron

Full Text Available Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

Science.gov (United States)

Theron, A; Roth, R L; Hoppe, H; Parkinson, C; van der Westhuyzen, C W; Stoychev, S; Wiid, I; Pietersen, R D; Baker, B; Kenyon, C P

2017-01-01

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

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Ana Rita Seabra

2015-07-01

Full Text Available Glutamine Synthetase (GS catalyses the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of glutamine synthetase isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

Equilibria and partitioning of complexes in the S-adenosylmethionine synthetase reaction

International Nuclear Information System (INIS)

Markham, G.D.

1987-01-01

S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a reaction in which the [enzyme-ATP-methionine] complex reacts to form an intermediate [enzyme-AdoMet-PPPi] complex: hydrolysis of PPPi yields an [enzyme-AdoMet-PPi-Pi] complex from which AdoMet is the last product to dissociate. Analysis of reaction mixtures which were quenched with acid during turnover of E. coli AdoMet synthetase with saturating substrates containing [α - 32 P]ATP showed that PPPi is present in an amount corresponding to 45% of the total enzyme active sites, reflecting the portion of enzyme present in an [enzyme-AdoMet-PPPi] complex. Similar experiments in which excess pyrophosphatase was included (to hydrolyze PPi as it was released from AdoMet synthetase), showed that enzyme-bound PPi is present in an amount corresponding to 22% of the total AdoMet synthetase. The enzyme not present in complexes with PPPi or PPi is probably distributed between the [enzyme-ATP-methionine] and the [enzyme-AdoMet] complexes. AdoMet synthetase forms enzyme-bound 32 PPPi from added 32 PPi and Pi; the equilibrium constant [enzyme-AdoMet-PPi-Pi]/[enzyme-AdoMet-PPPi] is 2.0, greatly displaced from the equilibrium for hydrolysis of free PPPi. Since the ratio of enzyme-bound PPi to PPPi is 0.5 during the steady state, the PPPi hydrolysis step is not at equilibrium during turnover. Formation of [ 32 P]ATP from the [enzyme-AdoMet- 32 PPPi] complex was not detected

The Inhibition of Folylpolyglutamate Synthetase (folC in the Prevention of Drug Resistance in Mycobacterium tuberculosis by Traditional Chinese Medicine

Directory of Open Access Journals (Sweden)

Tzu-Chieh Hung

2014-01-01

Full Text Available Tuberculosis (TB is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis.

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

Science.gov (United States)

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

2009-01-01

O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

The early history of tRNA recognition by aminoacyl-tRNA synthetases

Indian Academy of Sciences (India)

Madhu

2006-10-04

Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

Growth factors regulate glutamine synthetase activity in ...

African Journals Online (AJOL)

Khaled

2012-07-10

Jul 10, 2012 ... glutamate and ammonia, which in turn, cells are supplied with ammonia ... out to determine the maximum growth time at which cells will be .... Western blot technique for detection the glutamine synthetase enzyme. Lane 1;.

Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology

NARCIS (Netherlands)

Bischoff, Rainer; Kolbe, H.V.

1994-01-01

Non-enzymatic deamidation of asparagine and glutamine residues in proteins and peptides are reviewed by first outlining the well-described reaction mechanism involving cyclic imide intermediates, followed by a discussion of structural features which influence the reaction rate. The second and major

Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

Czech Academy of Sciences Publication Activity Database

Bakal, Tomáš; Goo, K.-S.; Najmanová, Lucie; Plháčková, Kamila; Kadlčík, Stanislav; Ulanová, Dana

2015-01-01

Roč. 108, č. 5 (2015), s. 1267-1274 ISSN 0003-6072 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Nonribosomal peptide synthetase * Adenylation domain * Actinomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 1.944, year: 2015

Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

Science.gov (United States)

Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

2003-01-01

The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

Radiation-induced G/sub 2/-arrest is reduced by inhibitors of poly(adenosine diphosphoribose) synthetase

International Nuclear Information System (INIS)

Rowley, R.

1985-01-01

Experiments are in progress to test whether poly(adenosine diphosphoribose) synthesis is required for the induction of G/sub 2/-arrest in growing mammalian cells following X-irradiation. A variety of poly(ADPR) synthetase inhibitors have been tested to determine: 1) whether addition of an inhibitor to X-irradiated CHO cells reduces G/sub 2/-arrest; 2) whether compounds structurally similar to poly-(ADPR) synthetase inhibitors but inactive against this enzyme affect radiation-induced G/sub 2/-arrest and 3) whether the concentration dependence for poly(ADPR) synthetase inhibition matches that for G/sub 2/-arrest reduction. G/sub 2/-arrest was measured in X-irradiated (1.5 Gy) CHO cells using the mitotic cell selection technique. Poly(ADPR) synthetase activity was measured in permeabilized cells by /sup 3/H-NAD incorporation. The synthetase inhibitors used were 3-aminobenzamide, benzamide, nicotinamide, 4-acetyl pyridine, caffeine and theophylline. The inactive compounds used were 3-aminobenzoic acid, benzoic acid, nicotinic acid, adenine, adenosine and 3'-deoxyadenosine. Inhibitors of poly(ADPR) synthetase reduced G/sub 2/-arrest while related compounds which produced no enzyme inhibition did not. The concentration dependencies for G/sub 2/-arrest reduction and enzyme inhibition were similar only for methyl xanthines. Further analysis awaits the determination of intracellular drug concentrations

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

Science.gov (United States)

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Measurement of Fructose-Asparagine Concentrations in Human and Animal Foods.

Science.gov (United States)

Wu, Jikang; Sabag-Daigle, Anice; Metz, Thomas O; Deatherage Kaiser, Brooke L; Gopalan, Venkat; Behrman, Edward J; Wysocki, Vicki H; Ahmer, Brian M M

2018-01-10

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as its sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the nonenzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules that contribute to the color, taste, and aroma of heated foods. Among these end derivatives is acrylamide, which is present in some foods, especially in fried potatoes. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 400 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have F-Asn at this level or higher (fresh apricots, lettuce, asparagus, and canned peaches). Much higher concentrations (11 000-35 000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report reveals possible origins of F-Asn as a nutrient source for Salmonella and identifies foods that could be used together with a FraB inhibitor as a therapeutic agent for Salmonella.

Measurement of fructose-asparagine concentrations in human and animal foods.

Energy Technology Data Exchange (ETDEWEB)

Wu, Jikang; Sabag-Daigle, Anice; Metz, Thomas O.; Kaiser, Brooke LD; Gopalan, Venkat; Behrman, Edward J.; Wysocki, Vicki H.; Ahmer, Brian

2018-01-10

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as the sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the non-enzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules which contribute to the color, taste, and aroma of heated foods. Among these is acrylamide, which is present in some foods, especially in fried potato products. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor, or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 399 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have this concentration or higher (fresh apricots, lettuce, asparagus, and in canned peaches). Much higher concentrations (11,000 to 35,000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report clarifies the origins of F-Asn as a nutrient source for Salmonella, and identifies foods that could be used in concert with a FraB inhibitor as a therapeutic agent for Salmonella.

Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

Energy Technology Data Exchange (ETDEWEB)

Nolan, N.L.; Kidwell, W.R.

1982-04-01

Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

International Nuclear Information System (INIS)

Nolan, N.L.; Kidwell, W.R.

1982-01-01

Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

NMR and spectroscopic studies on uranyl ion interaction with aspartic acid and asparagine

International Nuclear Information System (INIS)

Wieczorek, H.; Kozlowski, H.

1980-01-01

The carboxyl groups of peptides or proteins are quite effective in the binding of UO 2 +2 ion and as the first step in studies in that field aspartic acid has been chosen as the bi-carboxylic ligand. The data for UO 2 +2 -asparagine system are also presented in this communication as they complete the results obtained for the UO 2 +2 -aspartic acid system. (author)

L-Asparagine crystals with wide gap semiconductor features: optical absorption measurements and density functional theory computations.

Science.gov (United States)

Zanatta, G; Gottfried, C; Silva, A M; Caetano, E W S; Sales, F A M; Freire, V N

2014-03-28

Results of optical absorption measurements are presented together with calculated structural, electronic, and optical properties for the anhydrous monoclinic L-asparagine crystal. Density functional theory (DFT) within the generalized gradient approximation (GGA) including dispersion effects (TS, Grimme) was employed to perform the calculations. The optical absorption measurements revealed that the anhydrous monoclinic L-asparagine crystal is a wide band gap material with 4.95 eV main gap energy. DFT-GGA+TS simulations, on the other hand, produced structural parameters in very good agreement with X-ray data. The lattice parameter differences Δa, Δb, Δc between theory and experiment were as small as 0.020, 0.051, and 0.022 Å, respectively. The calculated band gap energy is smaller than the experimental data by about 15%, with a 4.23 eV indirect band gap corresponding to Z → Γ and Z → β transitions. Three other indirect band gaps of 4.30 eV, 4.32 eV, and 4.36 eV are assigned to α3 → Γ, α1 → Γ, and α2 → Γ transitions, respectively. Δ-sol computations, on the other hand, predict a main band gap of 5.00 eV, just 50 meV above the experimental value. Electronic wavefunctions mainly originating from O 2p-carboxyl, C 2p-side chain, and C 2p-carboxyl orbitals contribute most significantly to the highest valence and lowest conduction energy bands, respectively. By varying the lattice parameters from their converged equilibrium values, we show that the unit cell is less stiff along the b direction than for the a and c directions. Effective mass calculations suggest that hole transport behavior is more anisotropic than electron transport, but the mass values allow for some charge mobility except along a direction perpendicular to the molecular layers of L-asparagine which form the crystal, so anhydrous monoclinic L-asparagine crystals could behave as wide gap semiconductors. Finally, the calculations point to a high degree of optical

L-asparagine crystals with wide gap semiconductor features: Optical absorption measurements and density functional theory computations

Energy Technology Data Exchange (ETDEWEB)

Zanatta, G.; Gottfried, C. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, 90035-003 Porto Alegre-RS (Brazil); Silva, A. M. [Universidade Estadual do Piauí, 64260-000 Piripiri-Pi (Brazil); Caetano, E. W. S., E-mail: ewcaetano@gmail.com [Instituto de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza-CE (Brazil); Sales, F. A. M.; Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, Caixa Postal 6030, 60455-760 Fortaleza-CE (Brazil)

2014-03-28

Results of optical absorption measurements are presented together with calculated structural, electronic, and optical properties for the anhydrous monoclinic L-asparagine crystal. Density functional theory (DFT) within the generalized gradient approximation (GGA) including dispersion effects (TS, Grimme) was employed to perform the calculations. The optical absorption measurements revealed that the anhydrous monoclinic L-asparagine crystal is a wide band gap material with 4.95 eV main gap energy. DFT-GGA+TS simulations, on the other hand, produced structural parameters in very good agreement with X-ray data. The lattice parameter differences Δa, Δb, Δc between theory and experiment were as small as 0.020, 0.051, and 0.022 Å, respectively. The calculated band gap energy is smaller than the experimental data by about 15%, with a 4.23 eV indirect band gap corresponding to Z → Γ and Z → β transitions. Three other indirect band gaps of 4.30 eV, 4.32 eV, and 4.36 eV are assigned to α3 → Γ, α1 → Γ, and α2 → Γ transitions, respectively. Δ-sol computations, on the other hand, predict a main band gap of 5.00 eV, just 50 meV above the experimental value. Electronic wavefunctions mainly originating from O 2p–carboxyl, C 2p–side chain, and C 2p–carboxyl orbitals contribute most significantly to the highest valence and lowest conduction energy bands, respectively. By varying the lattice parameters from their converged equilibrium values, we show that the unit cell is less stiff along the b direction than for the a and c directions. Effective mass calculations suggest that hole transport behavior is more anisotropic than electron transport, but the mass values allow for some charge mobility except along a direction perpendicular to the molecular layers of L-asparagine which form the crystal, so anhydrous monoclinic L-asparagine crystals could behave as wide gap semiconductors. Finally, the calculations point to a high degree of optical

Characterization of arachidonate 5-lipoxygenase and leukotriene A4 synthetase from RBL-1 cells

International Nuclear Information System (INIS)

Cook, M.; Hogaboom, G.K.; Sarau, H.M.; Foley, J.J.; Crooke, S.T.

1986-01-01

5-lipoxygenase (LO) and leukotriene (LT) A4 synthetase from RBL-1 high speed (105,000 x g for 60 min) supernatants were partially purified by protein-high performance liquid chromatography (HPLC) and characterized in detail. The partially purified preparation contained only 5-LO and LTA4 synthetase and was isolated from 12-LO, peroxidase and LTA4 hydrolase activities. Reaction products were separated by reversed phase HPLC and quantitated by absorption spectrophotometry and radiochemical detection. The enzyme preparation rapidly converted [ 14 C]arachidonate to [ 14 C]5-hydroperoxyeicosatetraenoic acid (HPETE) and [ 14 C]5,12-dihydroperoxyeicosatetraenoic acids (diHETEs). The 5,12-diHETEs were primarily non-enzymatic breakdown products of LTA4 (e.g., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Both the 5-LO and LTA4 synthetase activities were Ca 2+- and ATP-dependent. For both enzyme activities, the CA 2+ stimulation required the presence of ATP. The fatty acid hydroperoxides, 5-,12-, and 15-HPETE, both stimulated ([ 3 μM]) 5-LO and LTA4 synthetase activities. The rapid isolation and subsequent characterization of 5-LO and LTA4 synthetase provide the bases for the further understanding of the role of the LO pathway in biological processes

A case of severe glutathione synthetase deficiency with novel GSS mutations

Science.gov (United States)

Xia, H.; Ye, J.; Wang, L.; Zhu, J.; He, Z.

2018-01-01

Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old. PMID:29340523

A case of severe glutathione synthetase deficiency with novel GSS mutations

Directory of Open Access Journals (Sweden)

H. Xia

2018-01-01

Full Text Available Glutathione synthetase deficiency (GSSD is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice

Directory of Open Access Journals (Sweden)

Xinghai Yang

2017-09-01

Full Text Available Nitrogen is a major nutritional element in rice production. However, excessive application of nitrogen fertilizer has caused severe environmental pollution. Therefore, development of rice varieties with improved nitrogen use efficiency (NUE is urgent for sustainable agriculture. In this study, bulked segregant analysis (BSA combined with whole genome re-sequencing (WGS technology was applied to finely map quantitative trait loci (QTL for NUE. A key QTL, designated as qNUE6 was identified on chromosome 6 and further validated by Insertion/Deletion (InDel marker-based substitutional mapping in recombinants from F2 population (NIL-13B4 × GH998. Forty-four genes were identified in this 266.5-kb region. According to detection and annotation analysis of variation sites, 39 genes with large-effect single-nucleotide polymorphisms (SNPs and large-effect InDels were selected as candidates and their expression levels were analyzed by qRT-PCR. Significant differences in the expression levels of LOC_Os06g15370 (peptide transporter PTR2 and LOC_Os06g15420 (asparagine synthetase were observed between two parents (Y11 and GH998. Phylogenetic analysis in Arabidopsis thaliana identified two closely related homologs, AT1G68570 (AtNPF3.1 and AT5G65010 (ASN2, which share 72.3 and 87.5% amino acid similarity with LOC_Os06g15370 and LOC_Os06g15420, respectively. Taken together, our results suggested that qNUE6 is a possible candidate gene for NUE in rice. The fine mapping and candidate gene analysis of qNUE6 provide the basis of molecular breeding for genetic improvement of rice varieties with high NUE, and lay the foundation for further cloning and functional analysis.

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

Directory of Open Access Journals (Sweden)

Tartar Aurélien

2010-06-01

Full Text Available Abstract Background Glutamine synthetase (GS is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria to the Chloroplastida. Results GSII sequences were isolated from four species of green algae (Trebouxiophyceae, and additional green algal (Chlorophyceae and Prasinophytae and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB and eukaryotic (GSIIE GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT. Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida. However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting

Improved stress tolerance and productivity in transgenic rice plants constitutively expressing the Oryza sativa glutathione synthetase OsGS under paddy field conditions.

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Park, Seong-Im; Kim, Young-Saeng; Kim, Jin-Ju; Mok, Ji-Eun; Kim, Yul-Ho; Park, Hyang-Mi; Kim, Il-Sup; Yoon, Ho-Sung

2017-08-01

Reactive oxygen species, which increase under various environmental stresses, have deleterious effects on plants. An important antioxidant, glutathione, is used to detoxify reactive oxygen species in plant cells and is mainly produced by two enzymes: gamma-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the glutathione synthetase gene (OsGS) in rice, we generated four independent transgenic rice plants (TG1-TG4) that overexpressed OsGS under the control of the constitutively expressed OsCc1 promoter. When grown under natural paddy field conditions, the TG rice plants exhibited greater growth development, higher chlorophyll content, and higher GSH/GSSH ratios than control wild-type (WT) rice plants. Subsequently, the TG rice plants enhanced redox homeostasis by preventing hydroperoxide-mediated membrane damage, which improved their adaptation to environmental stresses. As a result, TG rice plants improved rice grain yield and total biomass following increases in panicle number and number of spikelets per panicle, despite differences in climate during the cultivation periods of 2014 and 2015. Overall, our results indicate that OsGS overexpression improved redox homeostasis by enhancing the glutathione pool, which resulted in greater tolerance to environmental stresses in the paddy fields. Copyright © 2017. Published by Elsevier GmbH.

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

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Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

Thymidine kinase 2 and alanyl-tRNA synthetase 2 deficiencies cause lethal mitochondrial cardiomyopathy: case reports and review of the literature.

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Mazurova, Stella; Magner, Martin; Kucerova-Vidrova, Vendula; Vondrackova, Alzbeta; Stranecky, Viktor; Pristoupilova, Anna; Zamecnik, Josef; Hansikova, Hana; Zeman, Jiri; Tesarova, Marketa; Honzik, Tomas

2017-07-01

Cardiomyopathy is a common manifestation in neonates and infants with mitochondrial disorders. In this study, we report two cases manifesting with fatal mitochondrial hypertrophic cardiomyopathy, which include the third known patient with thymidine kinase 2 deficiency and the ninth patient with alanyl-tRNA synthetase 2 deficiency. The girl with thymidine kinase 2 deficiency had hypertrophic cardiomyopathy together with regression of gross motor development at the age of 13 months. Neurological symptoms and cardiac involvement progressed into severe myopathy, psychomotor arrest, and cardiorespiratory failure at the age of 22 months. The imaging methods and autoptic studies proved that she suffered from unique findings of leucoencephalopathy, severe, mainly cerebellar neuronal degeneration, and hepatic steatosis. The girl with alanyl-tRNA synthetase 2 deficiency presented with cardiac failure and underlying hypertrophic cardiomyopathy within 12 hours of life and subsequently died at 9 weeks of age. Muscle biopsy analyses demonstrated respiratory chain complex I and IV deficiencies, and histological evaluation revealed massive mitochondrial accumulation and cytochrome c oxidase-negative fibres in both cases. Exome sequencing in the first case revealed compound heterozygozity for one novel c.209T>C and one previously published c.416C>T mutation in the TK2 gene, whereas in the second case homozygozity for the previously described mutation c.1774C>T in the AARS2 gene was determined. The thymidine kinase 2 mutations resulted in severe mitochondrial DNA depletion (to 12% of controls) in the muscle. We present, for the first time, severe leucoencephalopathy and hepatic steatosis in a patient with thymidine kinase 2 deficiency and the finding of a ragged red fibre-like image in the muscle biopsy in a patient with alanyl-tRNA synthetase 2 deficiency.

The influence of prenatal X-irradiation on the activity of SRNA-aminoacyl synthetases in the developing rabbit brain

International Nuclear Information System (INIS)

Wender, M.; Zgorzalewicz, B.

1976-01-01

The activities of sRNA-aminoacyl synthetases were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single x-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. The results indicate clearly that prenatal exposure to ionizing radiation also affects the steps of protein biosynthesis which depend on the activity of sRNA-aminoacyl synthetases. (author)

An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

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Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D

2011-01-01

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translatio...... of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions....

Evolutionary anomalies among the aminoacyl-tRNA synthetases

Science.gov (United States)

Doolittle, R. F.; Handy, J.; Bada, J. L. (Principal Investigator)

1998-01-01

Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

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Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

1998-01-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction. PMID:9811660

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus.

Science.gov (United States)

Matussek, K; Moritz, P; Brunner, N; Eckerskorn, C; Hensel, R

1998-11-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.

Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

DEFF Research Database (Denmark)

Demant, Erland J.F.; Nystrøm, Birthe T.

2001-01-01

acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

Henoch-Schönlein purpura nephritis occurring postpartum in a patient with anti-PL-7 anti-synthetase syndrome.

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Nagai, Kojiro; Kishi, Jun; Morizumi, Shun; Minakuchi, Jun; Bando, Yoshimi; Nishioka, Yasuhiko; Doi, Toshio

2017-09-01

A 37-year-old pregnant woman developed purpura which was subsequently diagnosed as Henoch-Schönlein purpura (HSP). After childbirth, the patient developed proteinuria and hematuria. Further examination revealed that the HSP nephritis (HSPN) was associated with anti-threonyl-tRNA synthetase anti-synthetase syndrome. The onset of HSPN during pregnancy or after childbirth is rare. Moreover, to our knowledge, this is the first case to describe renal involvement in anti-synthetase syndrome.

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

International Nuclear Information System (INIS)

Chu, Wenchy; Horowitz, J.

1991-01-01

Interactions of 5-fluorouracil-substituted Escherichia coli tRNA Val with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K M and V max values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA Val . Binding of VRS to (FUra)tRNA Val induces structural perturbations that are reflected in selective changes in the 19 F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA Val along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA Val , suggesting conformational changes in this part of the molecule. No 19 F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA Val that has been proposed as a common intermediate in the aminoacylation reaction

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi.

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Byers, D M; Holmes, C G

1990-01-01

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

CSIR Research Space (South Africa)

Theron, Anjo

2017-10-01

Full Text Available mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does...

Stereospecific assignment of the NH2 resonances from the primary amides of asparagine and glutamine side chains in isotopically labeled proteins

International Nuclear Information System (INIS)

McIntosh, Lawrence P.; Brun, Emmanuel; Kay, Lewis E.

1997-01-01

An HMQC-based pulse scheme is presented for the stereospecific assignment of asparagine and glutamine side-chain amide protons. The approach makes use of the recently developed quantitative-J correlation spectroscopy [Bax, A. et al. (1994) Methods Enzymol., 239,79-105] to distinguish the E and Z primary amide protons and, as such, eliminates the need for assignments derived from more time-consuming and potentially ambiguous NOE methods. An application of this method to a uniformly 15N,13C-labeled cellulose-binding domain is presented. When used in combination with a NOESY-HSQC experiment, the predominant χ2 dihedral angles of two asparagine side chains in this protein can also be defined

Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

International Nuclear Information System (INIS)

Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A.

2009-01-01

Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals. FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P2 1 3, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively

Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions.

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Sharma, Richa; Jamwal, Vijaylakshmi; Singh, Varun P; Wazir, Priya; Awasthi, Praveen; Singh, Deepika; Vishwakarma, Ram A; Gandhi, Sumit G; Chaubey, Asha

2017-07-10

Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K + ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC-MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae. Copyright © 2017 Elsevier B.V. All rights reserved.

Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus

NARCIS (Netherlands)

Moorman, A. F.; de Boer, P. A.; Charles, R.; Lamers, W. H.

1990-01-01

A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary-treated rats was carried out, using radioactively-labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line

Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

Science.gov (United States)

Brattsten, L B; Wilkinson, C F

1975-07-01

1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.

A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.

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Christine Böttcher

Full Text Available An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-5'-[2-(1H-indol-3-ylethyl]phosphate (AIEP mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with K(i-values 17-68-fold lower than the respective K(m-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5-20 µM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

International Nuclear Information System (INIS)

Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

2007-01-01

DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K 2 ) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222 1 , with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

Rapid approach for cloning bacterial single-genes directly from soils ...

African Journals Online (AJOL)

Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

Stereospecific assignment of the asparagine and glutamine sidechain amide protons in proteins from chemical shift analysis

Energy Technology Data Exchange (ETDEWEB)

Harsch, Tobias; Schneider, Philipp; Kieninger, Bärbel; Donaubauer, Harald; Kalbitzer, Hans Robert, E-mail: hans-robert.kalbitzer@biologie.uni-regensburg.de [University of Regensburg, Institute of Biophysics and Physical Biochemistry and Centre of Magnetic Resonance in Chemistry and Biomedicine (Germany)

2017-02-15

Side chain amide protons of asparagine and glutamine residues in random-coil peptides are characterized by large chemical shift differences and can be stereospecifically assigned on the basis of their chemical shift values only. The bimodal chemical shift distributions stored in the biological magnetic resonance data bank (BMRB) do not allow such an assignment. However, an analysis of the BMRB shows, that a substantial part of all stored stereospecific assignments is not correct. We show here that in most cases stereospecific assignment can also be done for folded proteins using an unbiased artificial chemical shift data base (UACSB). For a separation of the chemical shifts of the two amide resonance lines with differences ≥0.40 ppm for asparagine and differences ≥0.42 ppm for glutamine, the downfield shifted resonance lines can be assigned to H{sup δ21} and H{sup ε21}, respectively, at a confidence level >95%. A classifier derived from UASCB can also be used to correct the BMRB data. The program tool AssignmentChecker implemented in AUREMOL calculates the Bayesian probability for a given stereospecific assignment and automatically corrects the assignments for a given list of chemical shifts.

The SH2D2A gene and susceptibility to multiple sclerosis

DEFF Research Database (Denmark)

Lorentzen, A.R.; Smestad, C.; Lie, B.A.

2008-01-01

We previously reported an association between the SH2D2A gene encoding TSAd and multiple sclerosis (MS). Here a total of 2128 Nordic MS patients and 2004 controls were genotyped for the SH2D2A promoter GA repeat polymorphism and rs926103 encoding a serine to asparagine substitution at amino acid...... that the SH2D2A gene may contribute to susceptibility to MS Udgivelsesdato: 2008/7/15...

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

International Nuclear Information System (INIS)

Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

2014-01-01

The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

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Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

2014-04-01

The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

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Zheng-xun JIN

2007-09-01

Full Text Available Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

Science.gov (United States)

Stephen, Preyesh; Ye, Sheng; Zhou, Ming; Song, Jian; Zhang, Rongguang; Wang, En-Duo; Giegé, Richard; Lin, Sheng-Xiang

2018-05-25

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Functional identification of glutamate cysteine ligase and glutathione synthetase in the marine yeast Rhodosporidium diobovatum

Science.gov (United States)

Kong, Min; Wang, Fengjuan; Tian, Liuying; Tang, Hui; Zhang, Liping

2018-02-01

Glutathione (GSH) fulfills a variety of metabolic functions, participates in oxidative stress response, and defends against toxic actions of heavy metals and xenobiotics. In this study, GSH was detected in Rhodosporidium diobovatum by high-performance liquid chromatography (HPLC). Then, two novel enzymes from R. diobovatum were characterized that convert glutamate, cysteine, and glycine into GSH. Based on reverse transcription PCR, we obtained the glutathione synthetase gene ( GSH2), 1866 bp, coding for a 56.6-kDa protein, and the glutamate cysteine ligase gene ( GSH1), 2469 bp, coding for a 90.5-kDa protein. The role of GSH1 and GSH2 for the biosynthesis of GSH in the marine yeast R. diobovatum was determined by deletions using the CRISPR-Cas9 nuclease system and enzymatic activity. These results also showed that GSH1 and GSH2 were involved in the production of GSH and are thus being potentially useful to engineer GSH pathways. Alternatively, pET- GSH constructed using vitro recombination could be used to detect the function of genes related to GSH biosynthesis. Finally, the fermentation parameters determined in the present study provide a reference for industrial GSH production in R. diobovatum.

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species.

Science.gov (United States)

Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Takahashi-Nakaguchi, Azusa; Matsuzawa, Tetsuhiro; Suzuki, Ken-ichiro; Fujita, Nobuyuki; Gonoi, Tohru

2014-04-30

Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology. Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text. We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

Science.gov (United States)

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

KAUST Repository

Li, Yongxin

2015-03-24

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

Science.gov (United States)

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

2015-03-01

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Unravelling the kinetics of the formation of acrylamide in the Maillard reaction of fructose and asparagine by multiresponse modelling

NARCIS (Netherlands)

Knol, J.J.; Linssen, J.P.H.; Boekel, van M.A.J.S.

2010-01-01

A kinetic model for the formation of acrylamide in a fructose–asparagine reaction system at initial pH 5.5 is proposed, based on an approach called multiresponse kinetic modelling. The formation of acetic acid and formic acid from the degradation of fructose and its isomer glucose was included in

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

Science.gov (United States)

Christensen, Karen E; Hou, Wenyang; Bahous, Renata H; Deng, Liyuan; Malysheva, Olga V; Arning, Erland; Bottiglieri, Teodoro; Caudill, Marie A; Jerome-Majewska, Loydie A; Rozen, Rima

2016-11-01

Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. Female Mthfd1S +/+ and Mthfd1S +/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S +/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater

Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

Science.gov (United States)

S.C. Minocha; R. Minocha; A. Komamine

1991-01-01

Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

Structural Analysis of the Active Site Geometry of N5-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli

International Nuclear Information System (INIS)

Thoden, James B.; Holden, Hazel M.; Firestine, Steven M.

2008-01-01

N 5 -Carboxyaminoimidazole ribonucleotide synthetase (N 5 -CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N 5 -CAIR, MgADP, and P i . The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N 5 -CAIR synthetase from Escherichia coli with either MgATP or MgADP/P i bound in the active site cleft. These structures, determined to 1.6-(angstrom) resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P i complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P i rather than ATP. The bound P i shifts by approximately 3 (angstrom) relative to the γ-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N 5 -CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N 5 -CAIR synthetase.

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

Science.gov (United States)

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-06-18

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

Energy Technology Data Exchange (ETDEWEB)

Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

2017-04-17

Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

International Nuclear Information System (INIS)

Baba, T.; Muth, J.; Allen, C.M.

1985-01-01

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [ 3 H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cations. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl 2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [ 14 C]isopentenyl pyrophosphate, and MgCl 2 , the labeled polypeptide gave a ratio of 14 C/ 3 H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cations are present

Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

International Nuclear Information System (INIS)

Guo, Min; Shapiro, Ryan; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization

Lack of protective effect of thromboxane synthetase inhibitor (CGS-13080) on single dose radiated canine intestine

International Nuclear Information System (INIS)

Barter, J.F.; Marlow, D.; Kamath, R.K.; Harbert, J.; Torrisi, J.R.; Barnes, W.A.; Potkul, R.K.; Newsome, J.T.; Delgado, G.

1991-01-01

The effect of a thromboxane A2 synthetase inhibitor (CGS-13080) on canine intestine was studied using a single dose of radiation, and radioactive microspheres were used to determine resultant blood flow. Thromboxane A2 causes vasospasm and platelet aggregation and may play a dominant role in radiation injury. However, there was no effect on the intestinal blood flow diminution occurring after radiation in this laboratory model using this thromboxane A2 synthetase inhibitor

STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

Science.gov (United States)

de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

2016-07-15

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

DEFF Research Database (Denmark)

Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

2011-01-01

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

Energy Technology Data Exchange (ETDEWEB)

Duan, Liangwei; Du, Jiansen [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Wang, Xuefeng; Zhou, Jianhua; Wang, Xiaojun [State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Liu, Xinqi, E-mail: liu2008@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2016-04-15

Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.

A liquid chromatography-tandem mass spectrometry method for simultaneous analysis of acrylamide and the precursors, asparagine and reducing sugars in bread

DEFF Research Database (Denmark)

Nielsen, N.J.; Granby, Kit; Hedegaard, Rikke Susanne Vingborg

2006-01-01

A LC-MS-MS method for simultaneous determination of acrylamide, asparagine, fructose, glucose and sucrose in bread was developed. The method is based on aqueous extraction by blending. After centrifugation the samples were cleaned up by solid phase extraction on C18 cartridges conditioned with 2 ...

Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

NARCIS (Netherlands)

He, Youji; Hakvoort, Theodorus B. M.; Vermeulen, Jacqueline L. M.; Labruyère, Wilhelmina T.; de Waart, D. Rudi; van der Hel, W. Saskia; Ruijter, Jan M.; Uylings, Harry B. M.; Lamers, Wouter H.

2010-01-01

Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/f)l mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in

Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

DEFF Research Database (Denmark)

Galbo, H; Saugmann, P; Richter, Erik

1979-01-01

Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

International Nuclear Information System (INIS)

Deschavanne, P.J.; Debieu, D.; Malaise, E.P.; Midander, J.; Revesz, L.

1986-01-01

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect. (author)

Seryl-tRNA Synthetases in Translation and Beyond

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Marko Močibob

2016-06-01

Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

DEFF Research Database (Denmark)

Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

2016-01-01

Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. 13C NMR and enzymatic assay

International Nuclear Information System (INIS)

Mesnard, F.; Marty, D.; Monti, J.P.; Gillet-Manceau, F.; Fliniaux, M.A.

1999-01-01

The metabolism of labelled pyruvate followed by 13 C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloid synthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

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Simona Kamenšek

2015-06-01

Full Text Available Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8, after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

SNP in Chalcone Synthase gene is associated with variation of 6-gingerol content in contrasting landraces of Zingiber officinale.Roscoe.

Science.gov (United States)

Ghosh, Subhabrata; Mandi, Swati Sen

2015-07-25

Zingiber officinale, medicinally the most important species within Zingiber genus, contains 6-gingerol as the active principle. This compound obtained from rhizomes of Z.officinale, has immense medicinal importance and is used in various herbal drug formulations. Our record of variation in content of this active principle, viz. 6-gingerol, in land races of this drug plant collected from different locations correlated with our Gene expression studies exhibiting high Chalcone Synthase gene (Chalcone Synthase is the rate limiting enzyme of 6-gingerol biosynthesis pathway) expression in high 6-gingerol containing landraces than in the low 6-gingerol containing landraces. Sequencing of Chalcone Synthase cDNA and subsequent multiple sequence alignment revealed seven SNPs between these contrasting genotypes. Converting this nucleotide sequence to amino acid sequence, alteration of two amino acids becomes evident; one amino acid change (asparagine to serine at position 336) is associated with base change (A→G) and another change (serine to leucine at position 142) is associated with the base change (C→T). Since asparagine at position 336 is one of the critical amino acids of the catalytic triad of Chalcone Synthase enzyme, responsible for substrate binding, our study suggests that landraces with a specific amino acid change viz. Asparagine (found in high 6-gingerol containing landraces) to serine causes low 6-gingerol content. This is probably due to a weak enzyme substrate association caused by the absence of asparagine in the catalytic triad. Detailed study of this finding could also help to understand molecular mechanism associated with variation in 6-gingerol content in Z.officinale genotypes and thereby strategies for developing elite genotypes containing high 6-gingerol content. Copyright © 2015 Elsevier B.V. All rights reserved.

The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae.

Science.gov (United States)

Persuhn, D C; Souza, E M; Steffens, M B; Pedrosa, F O; Yates, M G; Rigo, L U

2000-11-15

The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

DEFF Research Database (Denmark)

Willemoës, Martin; Hove-Jensen, Bjarne

1997-01-01

The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

Primary structure of the tms and prs genes of Bacillus subtilis

DEFF Research Database (Denmark)

Nilsson, Dan; Hove-Jensen, Bjarne; Arnvig, Kirsten

1989-01-01

The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene...... products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit Mr of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an Mr of 49,554. Comparison of the deduced B. subtilis PRPP...

Osmoregulation and nutritional relationships between Orobanche foetida and faba bean

Science.gov (United States)

Kharrat, Mohamed; Delavault, Philippe; Chaïbi, Wided; Simier, Philippe

2009-01-01

The present study aims at comparing the phloem composition of the tolerant XBJ90.03-16-1-1-1 and the susceptible Bachaar genotypes and the impact of the faba bean genotype on the levels of the major solutes and invertase activities in the parasite Orobanche foetida. In comparison to Bachaar, the XBJ90.03-161-1-1 genotype limited the growth of orobanche tubercles under in vitro conditions. The limited growth was due to low soluble invertase activity, low osmotic potential of the infected roots and the organic nitrogen deficiency of the host phloem sap. The faba bean genotype did not affect the osmoregulation process of O. foetida. Among the organic solutes, stachyose, hexoses, starch and free amino acids, mainly asparagine and aspartate were highly accumulated in orobanche. However, asparagine/aspartate, glutamine/glutamate, alanine, serine, gamma amino butyric acid, stachyose, sucrose were identified as the main organic components in the host phloem exudates. The key role of the enzymes α-galactosidase, asparagine synthetase and aspartate oxaloglutarate aminotransferase in the utilization of the host solutes is proposed in O. foetida parasitizing faba bean. PMID:19794856

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

OpenAIRE

Richaud, F; Richaud, C; Ratet, P; Patte, J C

1986-01-01

In Escherichia coli, the first enzyme of the diaminopimelate and lysine pathway is dihydrodipicolinate synthetase, which is feedback-inhibited by lysine and encoded by the dapA gene. The location of the dapA gene on the bacterial chromosome has been determined accurately with respect to the neighboring purC and dapE genes. The complete nucleotide sequence and the transcriptional start of the dapA gene were determined. The results show that dapA consists of a single cistron encoding a 292-amin...

Natural aminoacyl tRNA synthetase fragment enhances cardiac function after myocardial infarction.

Directory of Open Access Journals (Sweden)

Margaret E McCormick

Full Text Available A naturally-occurring fragment of tyrosyl-tRNA synthetase (TyrRS has been shown in higher eukaryotes to 'moonlight' as a pro-angiogenic cytokine in addition to its primary role in protein translation. Pro-angiogenic cytokines have previously been proposed to be promising therapeutic mechanisms for the treatment of myocardial infarction. Here, we show that systemic delivery of the natural fragment of TyRS, mini-TyrRS, improves heart function in mice after myocardial infarction. This improvement is associated with reduced formation of scar tissue, increased angiogenesis of cardiac capillaries, recruitment of c-kitpos cells and proliferation of myocardial fibroblasts. This work demonstrates that mini-TyrRS has beneficial effects on cardiac repair and regeneration and offers support for the notion that elucidation of the ever expanding repertoire of noncanonical functions of aminoacyl tRNA synthetases offers unique opportunities for development of novel therapeutics.

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine

DEFF Research Database (Denmark)

Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman

2004-01-01

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology......-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine....

Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

DEFF Research Database (Denmark)

Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

1970-01-01

- 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2....... A wide range (C4:0–C18:0) of fatty acids was synthesised and the proportions were modified by substrate concentrations in the same manner as for purified rabbit mammary gland fatty acid synthetase. - 3. The relative amount of radioactivity incorporated from added acetyl-CoA and malonyl-CoA depended...... of long-chain fatty acids was synthesised from carboxylated acetyl-CoA than from added malonyl-CoA. - 5. It is suggested that acetyl-CoA carboxylase may carboxylate acetate bound to fatty acid synthetase....

Holocarboxylase synthetase deficiency pre and post newborn screening

Directory of Open Access Journals (Sweden)

Taraka R. Donti

2016-06-01

Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Science.gov (United States)

Richaud, F; Richaud, C; Ratet, P; Patte, J C

1986-04-01

In Escherichia coli, the first enzyme of the diaminopimelate and lysine pathway is dihydrodipicolinate synthetase, which is feedback-inhibited by lysine and encoded by the dapA gene. The location of the dapA gene on the bacterial chromosome has been determined accurately with respect to the neighboring purC and dapE genes. The complete nucleotide sequence and the transcriptional start of the dapA gene were determined. The results show that dapA consists of a single cistron encoding a 292-amino acid polypeptide of 31,372 daltons.

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Science.gov (United States)

Richaud, F; Richaud, C; Ratet, P; Patte, J C

1986-01-01

In Escherichia coli, the first enzyme of the diaminopimelate and lysine pathway is dihydrodipicolinate synthetase, which is feedback-inhibited by lysine and encoded by the dapA gene. The location of the dapA gene on the bacterial chromosome has been determined accurately with respect to the neighboring purC and dapE genes. The complete nucleotide sequence and the transcriptional start of the dapA gene were determined. The results show that dapA consists of a single cistron encoding a 292-amino acid polypeptide of 31,372 daltons. Images PMID:3514578

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

Science.gov (United States)

Hadd, Andrew; Perona, John J

2014-12-19

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. {sup 13}C NMR and enzymatic assay; Activite de la glutamine synthetase dans des suspensions cellulaires de solanacees productrices ou non d'alcaloides. RMN du {sup 13}C et dosage enzymatique

Energy Technology Data Exchange (ETDEWEB)

Mesnard, F.; Marty, D.; Monti, J.P. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Biophysique, Groupe de Recherche des Biomolecules: micro-environnement et Metabolisme; Gillet-Manceau, F.; Fliniaux, M.A. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Phytotechnologie

1999-09-01

The metabolism of labelled pyruvate followed by {sup 13}C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloidsynthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

International Nuclear Information System (INIS)

Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

2011-01-01

Highlights: → Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). → Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. → HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

2016-01-15

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

Cancer association study of aminoacyl-tRNA synthetase signaling network in glioblastoma.

Directory of Open Access Journals (Sweden)

Yong-Wan Kim

Full Text Available Aminoacyl-tRNA synthetases (ARSs and ARS-interacting multifunctional proteins (AIMPs exhibit remarkable functional versatility beyond their catalytic activities in protein synthesis. Their non-canonical functions have been pathologically linked to cancers. Here we described our integrative genome-wide analysis of ARSs to show cancer-associated activities in glioblastoma multiforme (GBM, the most aggressive malignant primary brain tumor. We first selected 23 ARS/AIMPs (together referred to as ARSN, 124 cancer-associated druggable target genes (DTGs and 404 protein-protein interactors (PPIs of ARSs using NCI's cancer gene index. 254 GBM affymetrix microarray data in The Cancer Genome Atlas (TCGA were used to identify the probe sets whose expression were most strongly correlated with survival (Kaplan-Meier plots versus survival times, log-rank t-test <0.05. The analysis identified 122 probe sets as survival signatures, including 5 of ARSN (VARS, QARS, CARS, NARS, FARS, and 115 of DTGs and PPIs (PARD3, RXRB, ATP5C1, HSP90AA1, CD44, THRA, TRAF2, KRT10, MED12, etc. Of note, 61 survival-related probes were differentially expressed in three different prognosis subgroups in GBM patients and showed correlation with established prognosis markers such as age and phenotypic molecular signatures. CARS and FARS also showed significantly higher association with different molecular networks in GBM patients. Taken together, our findings demonstrate evidence for an ARSN biology-dominant contribution in the biology of GBM.

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

Science.gov (United States)

Hoepfner, Dominic; McNamara, Case W; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L; Plouffe, David M; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K; Petersen, Frank; Supek, Frantisek; Glynne, Richard J; Tallarico, John A; Porter, Jeffrey A; Fishman, Mark C; Bodenreider, Christophe; Diagana, Thierry T; Movva, N Rao; Winzeler, Elizabeth A

2012-06-14

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. Copyright © 2012 Elsevier Inc. All rights reserved.

Controlling the prion propensity of glutamine/asparagine-rich proteins.

Science.gov (United States)

Paul, Kacy R; Ross, Eric D

2015-01-01

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

Influence of endogenous pyrogen on the cerebral prostaglandin-synthetase system.

Science.gov (United States)

Ziel, R; Krupp, P

1976-11-15

The biotransformation of arachidonic acid to prostaglandins in vitro is specifically augmented by endogenous pyrogen to a degree depending on the concentration applied, providing that the microsomal fraction of the cerebral cortex is used as prostaglandin-synthetase system. This effect is inhibited by non-steroidal anti-inflammatory agents. These findings are compatible with the hypothesis that prostaglandins might act as mediators of the febrile reaction induced by endogenous pyrogen.

The functional properties of chitosan-glucose-asparagine Maillard reaction products and mitigation of acrylamide formation by chitosans.

Science.gov (United States)

Sung, Wen-Chieh; Chang, Yu-Wei; Chou, Yu-Hao; Hsiao, Hsin-I

2018-03-15

This research aims to clarify the interactions that occur in a food model system consisting of glucose, asparagine and chitosans. Low molecular weight chitosan exerted a potent inhibitory effect (46.8%) on acrylamide and Maillard reaction products (MRPs) (>52.6%), respectively. Compared to a previous study conducted using the fructose system, the novel findings of this research demonstrate that the formation of acrylamide and Maillard reaction products was lower with glucose than with fructose when they were used as reducing sugars in food model systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Nygaard, Per

1982-01-01

, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

OpenAIRE

Sanya Chadha; N. Arjunreddy Mallampudi; Debendra K. Mohapatra; Rentala Madhubala; Ira J. Blader; Greg Matlashewski; Frederick Buckner

2017-01-01

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L.?donovani lysyl-tRNA synthetase (LdLysRS). Two different codin...

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure Elucidation and Activity of Kolossin A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

Science.gov (United States)

Bode, Helge B; Brachmann, Alexander O; Jadhav, Kirtikumar B; Seyfarth, Lydia; Dauth, Christina; Fuchs, Sebastian W; Kaiser, Marcel; Waterfield, Nick R; Sack, Holger; Heinemann, Stefan H; Arndt, Hans-Dieter

2015-08-24

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.

Science.gov (United States)

Armen, Roger S; Schiller, Stefan M; Brooks, Charles L

2010-06-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

NARCIS (Netherlands)

Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.; Weiner, I. David

2016-01-01

Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of

Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

DEFF Research Database (Denmark)

Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

1983-01-01

The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

Regulations of enzymes in animals: effects of developmental processes, cancer and radiation. Progress report IX, 1 May 1974--31 April 1975

International Nuclear Information System (INIS)

Knox, W.E.

1975-01-01

Investigations of the properties of variant forms of emnzymes in rat tissues were continued. Two glutamyltransferases, one which remains associated with glutamine synthetase and the other which can be separated from it, were purified. A new assay method forglutaminase activity was established which facilitated further characterization of the 3 isozymes and their concentration in normal and neoplastic tissues. Studies of arginase led to the demonstration of the role that the new variant of arginase plays in proline synthesis in mammary gland. An inhibitor of asparagine synthetase, which is absent from fetal liver and tumors, was discovered in adult rat liver. Peptidyl proline hydroxylase (an essential enzyme in collagen synthesis) was identified as one of the most sensitive indicators of neoplastic growth. The spectrum of experimental, transplantable rat tumors was extended to a series of salivary gland tumors and a radiation-induced lymphoma. (U.S.)

Proofreading in vivo: Editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli

International Nuclear Information System (INIS)

Jakubowski, H.

1990-01-01

Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells

Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

Directory of Open Access Journals (Sweden)

Wei-Ching Chen

Full Text Available Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL

Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.

Science.gov (United States)

Khan, Sameena; Sharma, Arvind; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit

2014-06-01

Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

International Nuclear Information System (INIS)

Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A. S.; Macedo-Ribeiro, Sandra

2010-01-01

The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro

International Nuclear Information System (INIS)

Zhao Guohui; Liu Jun; Liu Xiang; Chen Min; Zhang Houcheng; Wang, Peng George

2007-01-01

GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K m and K cat /K m for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10 5 M -1 S -1 , and those for L-glutamate were 2 mM and 0.52 x 10 5 M -1 S -1 , respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7

Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

Directory of Open Access Journals (Sweden)

Jason M van Rooyen

Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

Science.gov (United States)

Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

2018-03-29

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium

DEFF Research Database (Denmark)

Hansen, Jørgen T.; Sørensen, Jens L.; Giese, Henriette

2012-01-01

Fusarium species produce a plethora of bioactive polyketides and nonribosomal peptides that give rise to health problems in animals and may have drug development potential. Using the genome sequences for Fusarium graminearum, F. oxysporum, F. solani and F. verticillioides we developed a framework...... and NRPS genes in sequenced Fusarium species and their known products. With the rapid increase in the number of sequenced fungal genomes a systematic classification will greatly aid the scientific community in obtaining an overview of the number of different NRPS and PKS genes and their potential...

Association of mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of Pol

Directory of Open Access Journals (Sweden)

Shalak V. F.

2011-10-01

Full Text Available Aim. Analyze the interaction between Lysyl-tRNA synthetase (LysRS and HIV-1 GagPol to know whether a particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol. Methods. Yeast two-hybrid analysis, immunoprecipitation. Results. We have shown that LysRS associates with the Pol domain of GagPol. Conclusions. A model of the assembly of the LysRS:tRNA3Lys:GagPol packaging complex is proposed.

Purification and properties of the dihydrofolate synthetase from Serratia indica

International Nuclear Information System (INIS)

Ikeda, Masamichi; Iwai, Kazuo

1976-01-01

The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg 2+ and K + as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum

Energy Technology Data Exchange (ETDEWEB)

Rydzak, Thomas [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Garcia, David [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Stevenson, David M. [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Sladek, Margaret [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Klingeman, Dawn M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Holwerda, Evert K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Amador-Noguez, Daniel [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Brown, Steven D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center

2017-05-01

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. And while recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H₂), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine and α-ketoglutarate levels indicative of nitrogen-rich conditions. Here, we propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine/α-ketoglutarate levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

Science.gov (United States)

Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

2014-03-01

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

DEFF Research Database (Denmark)

Noh, Soo Min; Shin, Seunghyeon; Min Lee, Gyun

2018-01-01

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1...... and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated...... in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation...

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Science.gov (United States)

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Directory of Open Access Journals (Sweden)

Cheryl Ingram-Smith

2006-01-01

Full Text Available Adenosine monophosphate (AMP-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming, EC 6.2.1.1 is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1 and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2, revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

DEFF Research Database (Denmark)

Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

1985-01-01

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosin...

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin: structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

International Nuclear Information System (INIS)

Green, E.D.; Baenziger, J.U.

1988-01-01

The authors have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[ 3 H] 4 . The 3 H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, they describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species

In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

Science.gov (United States)

Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

2016-05-01

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

A mutation in the MATP gene causes the cream coat colour in the horse

Directory of Open Access Journals (Sweden)

Guérin Gérard

2003-01-01

Full Text Available Abstract In horses, basic colours such as bay or chestnut may be partially diluted to buckskin and palomino, or extremely diluted to cream, a nearly white colour with pink skin and blue eyes. This dilution is expected to be controlled by one gene and we used both candidate gene and positional cloning strategies to identify the "cream mutation". A horse panel including reference colours was established and typed for different markers within or in the neighbourhood of two candidate genes. Our data suggest that the causal mutation, a G to A transition, is localised in exon 2 of the MATP gene leading to an aspartic acid to asparagine substitution in the encoded protein. This conserved mutation was also described in mice and humans, but not in medaka.

Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

DEFF Research Database (Denmark)

Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

2011-01-01

Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

Dissipation kinetics of asparagine in soil measured by compound-specific analysis with metabolite tracking

DEFF Research Database (Denmark)

Czaban, Weronika; Rasmussen, Jim; Nicolaisen, Mogens

2016-01-01

labeled glutamic acid were detected in soil. This highlights the fast turnover of amino acid in soil and that the estimation of concentration of the formed compounds is important when evaluating plant available organic N. Efficiency of the compound-specific analysis showed to be a powerful technique......Estimating the potential for direct plant acquisition of organic N, in particular amino acids, requires assessment of their turnover times in soil. It is well known from 14C studies that mineralization of amino acids occurs within hours, but mineralization to 14CO2 does not indicate the rate...... of disappearance of the intact amino acid or the possible formation of metabolites during amino acid dissipation. We here used compound-specific isotope analysis with metabolite tracking to investigate the dissipation rate of universally labeled intact 13C15N-asparagine at two concentrations and the subsequent...

Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

NARCIS (Netherlands)

Lamers, W. H.; van Roon, M.; Mooren, P. G.; de Graaf, A.; Charles, R.

1985-01-01

A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic

Adsorption of asparagine on the gold electrode and air/solution interface

International Nuclear Information System (INIS)

Slojkowska, R.; Palys, B.; Jurkiewicz-Herbich, M.

2004-01-01

The adsorption of asparagine (Asn) on a gold electrode from 0.1 M LiClO 4 aqueous solutions was investigated. The experimental data obtained from ac impedance measurements were analyzed to determine the dependence of adsorption parameters, i.e. the standard Gibbs energy of adsorption (ΔG 0 ), maximal value of surface excess concentration (Γ max ) of Asn and parameter of interactions in the adsorbed layer (A) on the electrode potential. The relatively large value of Gibbs energy of adsorption (∼ -47 kJ mol -1 ) gives the evidence of a very strong adsorption of Asn at the polycrystalline Au electrode. The comparison of the adsorption behavior of Asn at the air/solution and the Au/solution interfaces points out to the significant electronic interactions of adsorbate molecules with the Au electrode, since the adsorption of Asn on a free surface (from the same solutions) is very week. The analysis of the electrochemical data as well as the infrared reflection absorption spectroscopy (IRAS) results reveal that Asn molecules are anchored to the Au surface through oxygen atoms of the carboxylate group COO - and through the amide carbonyl group

Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

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Niran Roongsawang

2010-12-01

Full Text Available Lipopeptide biosurfactants (LPBSs consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive and Pseudomonas (Gram-negative have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs. Production of active‑form NRPSs requires not only transcriptional induction and translation but also post‑translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

Aminoacyl-tRNA synthetases database Y2K.

Science.gov (United States)

Szymanski, M; Barciszewski, J

2000-01-01

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html

The effect of Cu{sup II} ions in L-asparagine single crystals

Energy Technology Data Exchange (ETDEWEB)

Santana, Ricardo C., E-mail: santana@ufg.br; Gontijo, Henrique O.; Menezes, Arthur F.; Martins, José A.; Carvalho, Jesiel F., E-mail: carvalho@ufg.br

2016-11-15

We report the synthesis, crystal growth, and spectroscopic characterization of L-asparagine monohydrate (LAM) single crystals doped with CuII. The crystals were successfully grown by slow cooling from a supersaturated aqueous solution up to size of 16×12×2 mm{sup 3};the effect of copper impurities in the crystals morphology was discussed. Electron Paramagnetic Resonance (EPR) was used to calculate the g and hyperfine coupling (A) tensors of the CuII ions (g{sub 1}=2.044, g{sub 2}=2.105, g{sub 3}=2.383and A{sub 1}≈0, A{sub 2}=35, A{sub 3}=108 Gauss). The EPR spectra for certain orientations of the magnetic field suggest that CuII ions are coordinated to two {sup 14}N atoms. Correlating the EPR and optical absorption results, the crystal field and the Cu{sup II} orbital bond parameters were calculated. The results indicate that the paramagnetic center occupies interstitial rhombic distorted site and the ground orbital state for the unpaired electron is the d(x{sup 2}-y{sup 2}).

Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer

Science.gov (United States)

SHAN, YAN-SHEN; HSU, HUI-PING; LAI, MING-DERG; YEN, MENG-CHI; LUO, YI-PEY; CHEN, YI-LING

2015-01-01

Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin-embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer. PMID:25333458

Kinetic isotope effect studies of the S-adenosylmethionine synthetase reaction

International Nuclear Information System (INIS)

Markham, G.D.; Parkin, D.W.; Schramm, V.L.

1986-01-01

S-adenosylmethionine (AdoMet) synthetase catalyzes a unique substitution reaction at the 5' carbon of MgATP. Kinetic isotope effect (V/K) measurements have been used to investigate the mechanism of AdoMet synthetase from E. coli. Changes in 3 H/ 14 C ratios when AdoMet is formed from a mixture of either ([5'- 14 C]ATP and [5'- 12 C,1'- 3 H]ATP) or ([5'- 3 H]ATP and [5'- 1 H,1'- 14 C]ATP) were examined. The effects of varying the concentrations of the co-substrate methionine and the monovalent cation activator K + were investigated. Substitution of 14 C for 12 C at the 5' position of ATP yields a primary V/K kinetic isotope effect ( 12 C/ 14 C) of 1.128 +/- 0.004 at low K + and methionine concentrations. The observed isotope effect diminishes slightly to 1.107 +/- 0.003 when both K + and methionine are present at saturating concentrations, suggesting that MgATP has only a low commitment to catalysis from at conditions near Vmax. No secondary V/K 3 H isotope effect from [5'- 3 H]ATP was detected ( 1 H/ 3 H) = 0.997 +/- 0.003. The magnitude of the primary 14 C isotope effect and the small secondary 3 H effect demonstrate that AdoMet synthesis occurs with a S/sub N/ 2 transition state which is symmetric with respect to the sulfur nucleophile and the departing tripolyphosphate group

Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production in Streptomyces coelicolor A3(2)

NARCIS (Netherlands)

D'Alia, Davide; Nieselt, Kay; Steigele, Stephan; Mueller, Jonas; Verburg, Ilse; Takano, Eriko; Alia, Davide D’; Müller, Jonas

Overexpression of antisense chromosomal cis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production in Streptomyces coelicolor. In addition, we predicted 3,597 cis-encoded ncRNAs and validated 13 of them

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin: distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

International Nuclear Information System (INIS)

Green, E.D.; Baenziger, J.U.

1988-01-01

The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In this study, the authors determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear α-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by >10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (α2,3 versus α2,6). The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity

The Disappearance of a Hepatic Mass in Anti-Synthetase Syndrome

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Christopher J Mesa

2017-06-01

Full Text Available Anti-Synthetase Syndrome (ASyS is a rare chronic autoimmune disorder characterized by myositis, interstitial lung disease (ILD, polyarthralgia, “mechanic’s hands” and Raynaud’s phenomenon. Liver lesions are quite rare in ASyS. In our ASyS case, we will discuss a 58-year-old man presenting with muscle weakness, arthralgia, and interstitial lung disease (ILD. He was positive for anti-Jo-1 antibodies, substantiating the diagnosis, and was started on treatment. This was followed by the appearance of a liver mass that disappeared when the patient achieved remission.

Cloning and molecular analysis of L-asparaginase II gene (ansB

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ZEINAT K. MOHAMED

2015-12-01

Full Text Available The deamination of L-asparagine to L-aspartic acid and ammonia is catalyzed by L-asparaginases (L-asparagine amino hydrolase. The enzyme L-asparaginase is widely distributed in nature from different living organisms, starting from bacteria till mammals and plants. It has been recently thought to be a therapeutic agent in treatment of various lymphoblastic leukemia diseases. There have been many attempts to isolate microorganisms that produce L-asparaginase. L-ASNase producing bacteria, Escherichia coli MG27, was previously isolated from the River Nile and identified. In this study, ansB gene, encoding L-ASNase II from E. coli MG27, was amplified by PCR, cloned and characterized by DNA sequencing. The DNA sequence was then analyzed using bioinformatics analysis and translated into amino acid sequence. Identification of highly conserved amino acid sequence motifs was conducted by comparison against the InterPro database. Analysis revealed that the protein sequence had a catalytic domain of L-asparaginase type II (IPR004550 that belong to asparaginase/glutaminase family (IPR006034 and has asparaginase/glutaminase conserved site (IPR020827. According to results predicted using PSIpred tool, ansB consists of eight α-helices and 13 β-strands.

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

Science.gov (United States)

Rydzak, Thomas; Garcia, David; Stevenson, David M; Sladek, Margaret; Klingeman, Dawn M; Holwerda, Evert K; Amador-Noguez, Daniel; Brown, Steven D; Guss, Adam M

2017-05-01

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H 2 ), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum. Copyright © 2017. Published by Elsevier Inc.

Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii

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Qian Chao-Dong

2012-09-01

Full Text Available Abstract Background Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a β-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. Results A potential pelgipeptin synthetase gene cluster (plp was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs, with one, seven, and one module(s, respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1 provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. Conclusions In this study, a gene cluster (plp responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine.

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Mohamed M Ali

2014-06-01

Full Text Available Salmonella enterica serovar Typhimurium (Salmonella is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn, which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10-/- mice. The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively. The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.

Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy

Science.gov (United States)

Zhao, Z.; Hashiguchi, A.; Sakiyama, Y.; Okamoto, Y.; Tokunaga, S.; Zhu, L.; Shen, H.; Takashima, H.

2012-01-01

Objective: To identify a new genetic cause of distal hereditary motor neuropathy (dHMN), which is also known as a variant of Charcot-Marie-Tooth disease (CMT), in a Chinese family. Methods: We investigated a Chinese family with dHMN clinically, electrophysiologically, and genetically. We screened for the mutations of 28 CMT or related pathogenic genes using an originally designed microarray resequencing DNA chip. Results: Investigation of the family history revealed an autosomal dominant transmission pattern. The clinical features of the family included mild weakness and wasting of the distal muscles of the lower limb and foot deformity, without clinical sensory involvement. Electrophysiologic studies revealed motor neuropathy. MRI of the lower limbs showed accentuated fatty infiltration of the gastrocnemius and vastus lateralis muscles. All 4 affected family members had a heterozygous missense mutation c.2677G>A (p.D893N) of alanyl-tRNA synthetase (AARS), which was not found in the 4 unaffected members and control subjects. Conclusion: An AARS mutation caused dHMN in a Chinese family. AARS mutations result in not only a CMT phenotype but also a dHMN phenotype. PMID:22573628

Digestion of a single meal affects gene expression of ion and ammonia transporters and glutamine synthetase activity in the gastrointestinal tract of freshwater rainbow trout.

Science.gov (United States)

Bucking, Carol; Wood, Chris M

2012-04-01

Experiments on freshwater rainbow trout, Oncorhynchus mykiss, demonstrated how digestion affected the transcriptional expression of gastrointestinal transporters following a single satiating meal (~3% body mass ration) after a 1-week fast. Quantitative real-time polymerase chain reaction was employed to measure the relative mRNA expression of three previously cloned and sequenced transporters [H(+)-K(+)-ATPase (HKA), Na(+)/HCO(3)(-) cotransporter (NBC), and the Rhesus glycoprotein (Rhbg1; an ammonia transporter)] over a 24-h time course following feeding. Plasma total ammonia increased about threefold from pre-feeding levels to 288 μmol l(-1), whereas total ammonia levels in chyme supernatant reached a sixfold higher value (1.8 mmol l(-1)) than plasma levels. Feeding did not appear to have a statistically significant effect on the relative mRNA expression of the gastric HKA or Rhbg1. However, the relative mRNA expression of gastric NBC was increased 24 h following the ingestion of a meal. Along the intestinal tract, feeding increased the relative mRNA expression of Rhbg1, but had no effect on the expression of NBC. Expression of the gastric HKA was undetectable in the intestinal tract of freshwater rainbow trout. Digestion increased the activity of glutamine synthetase in the posterior intestine at 12 and 24 h following feeding. This study is among the first to show that there are digestion-associated changes in gene expression and enzyme activity in the gastrointestinal tract of teleost fish illustrating the dynamic plasticity of this organ. These post-prandial changes occur over the relative short-term duration of digesting a single meal.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

International Nuclear Information System (INIS)

Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

2007-01-01

The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55, b = 90, c = 96 Å

Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Waard, P. de; Koorevaar, A.; Kamerling, J.P.

1991-01-01

The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F (PNGase- F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and

Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

Science.gov (United States)

Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

2016-06-27

This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The better growth phenotype of DvGS1-transgenic arabidopsis thaliana is attributed to the improved efficiency of nitrogen assimilation

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Zhu Chenguang

2015-01-01

Full Text Available The overexpression of the algal glutamine synthetase (GS gene DvGS1 in Arabidopsis thaliana resulted in higher plant biomass and better growth phenotype. The purpose of this study was to recognize the biological mechanism for the growth improvement of DvGS1-transgenic Arabidopsis. A series of molecular and biochemical investigations related to nitrogen and carbon metabolism in the DvGS1-transgenic line was conducted. Analysis of nitrogen use efficiency (NUE-related gene transcription and enzymatic activity revealed that the transcriptional level and enzymatic activity of the genes encoding GS, glutamate synthase, glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, were significantly upregulated, especially from leaf tissues of the DvGS1-transgenic line under two nitrate conditions. The DvGS1-transgenic line showed increased total nitrogen content and decreased carbon: nitrogen ratio compared to wild-type plants. Significant reduced concentrations of free nitrate, ammonium, sucrose, glucose and starch, together with higher concentrations of total amino acids, individual amino acids (glutamate, aspartate, asparagine, methionine, soluble proteins and fructose in leaf tissues confirmed that the DvGS1-transgenic line demonstrated a higher efficiency of nitrogen assimilation, which subsequently affected carbon metabolism. These improved metabolisms of nitrogen and carbon conferred the DvGS1-transgenic Arabidopsis higher NUE, more biomass and better growth phenotype compared with the wild-type plants.

Targeting CPS1 in the treatment of Carbamoyl phosphate synthetase 1 (CPS1) deficiency, a urea cycle disorder.

Science.gov (United States)

Diez-Fernandez, Carmen; Häberle, Johannes

2017-04-01

Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder (UCD), which can lead to life-threatening hyperammonemia. Unless promptly treated, it can result in encephalopathy, coma and death, or intellectual disability in surviving patients. Over recent decades, therapies for CPS1D have barely improved leaving the management of these patients largely unchanged. Additionally, in many cases, current management (protein-restriction and supplementation with citrulline and/or arginine and ammonia scavengers) is insufficient for achieving metabolic stability, highlighting the importance of developing alternative therapeutic approaches. Areas covered: After describing UCDs and CPS1D, we give an overview of the structure- function of CPS1. We then describe current management and potential novel treatments including N-carbamoyl-L-glutamate (NCG), pharmacological chaperones, and gene therapy to treat hyperammonemia. Expert opinion: Probably, the first novel CPS1D therapies to reach the clinics will be the already commercial substance NCG, which is the standard treatment for N-acetylglutamate synthase deficiency and has been proven to rescue specific CPS1D mutations. Pharmacological chaperones and gene therapy are under development too, but these two technologies still have key challenges to be overcome. In addition, current experimental therapies will hopefully add further treatment options.

Targeting Prolyl-tRNA Synthetase to Accelerate Drug Discovery against Malaria, Leishmaniasis, Toxoplasmosis, Cryptosporidiosis, and Coccidiosis.

Science.gov (United States)

Jain, Vitul; Yogavel, Manickam; Kikuchi, Haruhisa; Oshima, Yoshiteru; Hariguchi, Norimitsu; Matsumoto, Makoto; Goel, Preeti; Touquet, Bastien; Jumani, Rajiv S; Tacchini-Cottier, Fabienne; Harlos, Karl; Huston, Christopher D; Hakimi, Mohamed-Ali; Sharma, Amit

2017-10-03

Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

DEFF Research Database (Denmark)

Paul, David S; Grevengoed, Trisha J; Pascual, Florencia

2014-01-01

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1(H-/-)), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy...... and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1(H-/-) hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1(H......-/-) hearts exhibited an 8-fold higher uptake of 2-deoxy[1-(14)C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-(14)C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1(H...

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

NARCIS (Netherlands)

Lamers, W. H.; Zonneveld, D.; Charles, R.

1984-01-01

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

Science.gov (United States)

Bushley, Kathryn E.; Raja, Rajani; Jaiswal, Pankaj; Cumbie, Jason S.; Nonogaki, Mariko; Boyd, Alexander E.; Owensby, C. Alisha; Knaus, Brian J.; Elser, Justin; Miller, Daniel; Di, Yanming; McPhail, Kerry L.; Spatafora, Joseph W.

2013-01-01

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role

Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

NARCIS (Netherlands)

Dubern, J.F.; Coppoolse, E.R.; Stiekema, W.J.; Bloemberg, G.V.

2008-01-01

Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene

Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

International Nuclear Information System (INIS)

Safford, R.; de Silva, J.; Lucas, C.

1987-01-01

Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from ∼ 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH

Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

Directory of Open Access Journals (Sweden)

Adam C. Mirando

2014-12-01

Full Text Available In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

Science.gov (United States)

Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

2014-04-04

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

Redox status affects the catalytic activity of glutamyl-tRNA synthetase

DEFF Research Database (Denmark)

Katz, Assaf; Banerjee, Rajat; de Armas, Merly

2010-01-01

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in t...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

Severe respiratory failure as a presenting feature of an interstitial lung disease associated with anti-synthetase syndrome (ASS).

Science.gov (United States)

Piroddi, Ines Maria Grazia; Ferraioli, Gianluca; Barlascini, Cornelius; Castagneto, Corrado; Nicolini, Antonello

2016-07-01

Anti-synthetase syndrome (ASS) is defined as a heterogeneous connective tissue disorder characterized by the association of an interstitial lung disease (ILD) with or without inflammatory myositis with the presence of anti-aminoacyl-tRNA-synthetase antibodies. ILD is one of the major extra-muscular manifestations of polymyositis and dermatomyositis. We report a case of a patient with dyspnea, cough, and intermittent fever as well as ILD associated ASS in the absence of muscular involvement. This patient was admitted to the emergency department with severe respiratory failure requiring non-invasive ventilation. Our patient's case demonstrates that the diagnosis of ASS may not be obvious. However, its diagnosis leads to appropriate and potentially life-saving treatment. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

International Nuclear Information System (INIS)

Joyce, Michael A.; Brownie, Edward R.; Hayakawa, Koto; Fraser, Marie E.

2007-01-01

Attempts to crystallize succinyl-CoA synthetase from the thermophile T. aquaticus were thwarted by proteolysis of the β-subunit and preferential crystallization of a truncated form. Crystals of the full-length enzyme were grown after the purification protocol was modified to include frequent additions of protease inhibitors. Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the β-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 Å resolution, are of the protein in complex with Mn 2+ -GDP

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

Science.gov (United States)

Klein, H. P.; Jahnke, L.

1979-01-01

Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

Essential nontranslational functions of tRNA synthetases.

Science.gov (United States)

Guo, Min; Schimmel, Paul

2013-03-01

Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. Although these new functions were thought to be 'moonlighting activities', many are as critical for cellular homeostasis as their activity in translation. New roles have been associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect pathways for cardiovascular development and the immune response and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. New architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. Although a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid-binding site for another purpose.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

Energy Technology Data Exchange (ETDEWEB)

Levin, Inna; Kessler, Naama [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Moor, Nina [Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk (Russian Federation); Klipcan, Liron [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Koc, Emine [Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 (United States); Templeton, Paul [Department Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215 (United States); Spremulli, Linda [Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 (United States); Safro, Mark, E-mail: mark.safro@weizmann.ac.il [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

2007-09-01

The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

Science.gov (United States)

Khan, Sameena; Garg, Ankur; Camacho, Noelia; Van Rooyen, Jason; Kumar Pole, Anil; Belrhali, Hassan; Ribas de Pouplana, Lluis; Sharma, Vinay; Sharma, Amit

2013-05-01

Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively.

Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation.

Science.gov (United States)

Na, Jiwoon; Jung, Jisu; Bang, Jeyoung; Lu, Qiao; Carlson, Bradley A; Guo, Xiong; Gladyshev, Vadim N; Kim, Jinhong; Hatfield, Dolph L; Lee, Byeong Jae

2018-04-30

Selenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or Drosophila leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the

A novel AAT-deletion mutation in the coding sequence of the BCO2 gene in yellow-fat rabbits.

Science.gov (United States)

Strychalski, Janusz; Brym, Paweł; Czarnik, Urszula; Gugołek, Andrzej

2015-11-01

The carcasses of yellow-fat rabbits may be attractive to modern consumers, because they have a relatively high content of biologically active compounds. One of the main candidate genes associated with the yellow-fat trait is β-carotene 9',10'-oxygenase (BCO2). This study is the first report of the novel AAT-deletion mutation at codon 248 of the BCO2 gene, which has been found in homozygous yellow-fat rabbits. The deletion mutation, located at the beginning of exon 6, results in the absence of asparagine in protein. We also developed a PCR-RFLP test that supports intravital genotyping of indel polymorphism based on genomic DNA.

Anti-synthetase syndrome associated with anti PL-12 and anti-Signal recognition particle antibodies and a necrotizing auto-immune myositis.

Science.gov (United States)

Malkan, Ashish; Cappelen-Smith, Cecilia; Beran, Roy; Griffith, Neil; Toong, Catherine; Wang, Min-Xia; Cordato, Dennis

2015-02-01

We report a 37-year-old woman with a 2 month history of proximal muscle weakness and extremely high creatine kinase (21,808 U/L) due to necrotizing auto-immune myositis (NAM) in association with anti-synthetase syndrome. Myositis-specific auto-immune antibody panel was positive for anti-Signal recognition particle and anti-PL-12. CT scan of the chest confirmed interstitial lung disease. Prednisolone, intravenous immunoglobulin and cyclophosphamide therapy was given with gradual improvement. This patient is notable for the unusual combination of NAM and anti-synthetase syndrome with the rare finding of two myositis-specific autoantibodies, which directed testing for associated extramuscular features and management with more aggressive immunotherapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment

OpenAIRE

Saint-L?ger, Ad?la?de; Sinadinos, Christopher; Ribas de Pouplana, Llu?s

2016-01-01

Malaria remains a major global health problem. Parasite resistance to existing drugs makes development of new antimalarials an urgency. The protein synthesis machinery is an excellent target for the development of new anti-infectives, and aminoacyl-tRNA synthetases (aaRS) have been validated as antimalarial drug targets. However, avoiding the emergence of drug resistance and improving selectivity to target aaRS in apicomplexan parasites, such as Plasmodium falciparum, remain crucial challenge...

Calcium regulates the expression of a Dictyostelium discoideum ...

Indian Academy of Sciences (India)

In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by ...

Think big--giant genes in bacteria.

Science.gov (United States)

Reva, Oleg; Tümmler, Burkhard

2008-03-01

Long genes should be rare in archaea and eubacteria because of the demanding costs of time and resources for protein production. The search in 580 sequenced prokaryotic genomes, however, revealed 0.2% of all genes to be longer than 5 kb (absolute number: 3732 genes). Eighty giant bacterial genes of more than 20 kb in length were identified in 47 taxa that belong to the phyla Thermotogae (1), Chlorobi (3), Planctomycetes (1), Cyanobacteria (2), Firmicutes (7), Actinobacteria (9), Proteobacteria (23) or Euryarchaeota (1) (number of taxa in brackets). Giant genes are strain-specific, differ in their tetranucleotide usage from the bulk genome and occur preferentially in non-pathogenic environmental bacteria. The two longest bacterial genes known to date were detected in the green sulfur bacterium Chlorobium chlorochromatii CaD3 encoding proteins of 36 806 and 20 647 amino acids, being surpassed in length only by the human titin coding sequence. More than 90% of bacterial giant genes either encode a surface protein or a polyketide/non-ribosomal peptide synthetase. Most surface proteins are acidic, threonine-rich, lack cystein and harbour multiple amino acid repeats. Giant proteins increase bacterial fitness by the production of either weapons towards or shields against animate competitors or hostile environments.

Mitigation of the processing contaminant acrylamide in bread by reducing asparagine in the bread dough.

Science.gov (United States)

Katsaiti, Tatiana; Granby, Kit

2016-09-01

Over the past few years there has been an increasing awareness regarding acrylamide (AAM) content of various foods. Although there are several relevant articles on AAM mitigation in industrially prepared products, the literature regarding homemade preparations is rather scarce. The objective of this study is to mitigate the AAM formation in baked buns made with 1:1 sifted wheat/wholegrain flour through the depletion of asparagine (ASN) in the bread dough. Using a full-factorial design, the effect of four factors (yeast amount, fermentation time, fermentation temperature and yeast types) was tested. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for AAM and its main precursor, ASN, determination. The resulting ASN depletion in the dough (68-89%) is significantly affected by fermentation time and yeast type, while AAM mitigation levels in the baked buns are significantly influenced by yeast amount, fermentation time and yeast type. The mean concentrations for each combination range between 5 and 15 µg kg(-)(1).

Face-selective crystal growth behavior of L-aspartic acid in the presence of L-asparagine

Science.gov (United States)

Sato, Hiroyasu; Doki, Norihito; Yoshida, Saki; Yokota, Masaaki; Shimizu, Kenji

2016-02-01

The kinetic mechanism of L-asparagine (L-Asn) action on L-aspartic acid (L-Asp) crystal growth, namely the face-selective effect of L-Asn on the L-Asp crystal growth rate in each direction, was examined. In the a-axis direction, the effect of L-Asn on the L-Asp crystal growth rate was small. Enhancement and inhibition of L-Asp crystal growth, and interestingly the dissolution of the L-Asp crystal face, were observed in the b-axis direction, depending on the amount of L-Asn added. In the c-axis direction, the L-Asp crystal growth rate decreased with the increase in the amount of L-Asn added, and the experimental results were well fitted with a Langmuir adsorption isotherm. The study showed that there were crystal growth conditions where enhancement and inhibition, as well as inhibition and dissolution, coexisted in the presence of an additive with a structure similar to the growing crystal.

Enhancement of lysyl-tRNA synthetase activity in the Enterobacteriaceae

International Nuclear Information System (INIS)

Hickey, E.W.; Hirshfield, I.

1987-01-01

Lysyl-tRNA synthetase (LRS) in E. coli is coded by two genes, one constitutive, and the other inducible; the latter is a cell stress protein. To determine if this system is wide spread in prokaryotes, the inducibility of LRS was first tested in eight members of the Enterobacteriaceae using cultural conditions known to induce the enzyme in E. coli K-12. Uninduced control cultures were grown to an O.D. of 0.2 at 580 nm in a supplemented minimal medium (SMM), pH 7.0 at 37 0 C. Induction stimuli include: growth in SMM with 3mM Gly-L-Leu; growth in SMM as above, but with the initial pH adjusted to 5.0; or growth in Difco AC Broth to early stationary phase with a concomitant drop in the pH of the medium below 5.5. LRS activity was assayed in whole-cell sonic extracts by the aminoacylation of crude E. coli tRNA by 14 C-lysine at pH 7.8 for three minutes. When E. aerogenes, K. pneumoniae, C. freundii, and S. typhimurium were grown in AC Broth, LRS activity was enhanced 2 to 4 fold. The enzyme is induced 2 to 4 fold in C. freundii and S. typhimurium upon growth at pH 5.0, whereas E. coli, K.; pneumoniae, and E. aerogenes show only a 1.5 fold induction. The peptide Gly-L-Leu enhanced LRS activity only in E. coli. LRS was not found to be inducible in S. marcescens, M. morganii, P. mirabilis, or P. vulgaris by any of the stimuli

Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

Science.gov (United States)

Zheng, Desen; Burr, Thomas J

2016-02-01

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

International Nuclear Information System (INIS)

Iwaki, Jun; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Hasegawa, Tsunemi

2005-01-01

Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K. Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K

Glutamine Synthetase: Localization Dictates Outcome

Directory of Open Access Journals (Sweden)

Alessandra Castegna

2018-02-01

Full Text Available Glutamine synthetase (GS is the adenosine triphosphate (ATP-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event. In vitro evidence, confirmed in vivo in some cases, suggests that reduced GS activity in cancer cells associates with a more invasive and aggressive phenotype. However, GS is known to be highly expressed in cells of the tumor microenvironment, such as fibroblasts, adipocytes and immune cells, and their ability to synthesize glutamine is responsible for the acquisition of protumoral phenotypes. This has opened a new window into the complex scenario of the tumor microenvironment, in which the balance of glutamine consumption versus glutamine synthesis influences cellular function. Since GS expression responds to glutamine starvation, a lower glutamine synthesizing power due to the absence of GS in cancer cells might apply a metabolic pressure on stromal cells. This event might push stroma towards a GS-high/protumoral phenotype. When referred to stromal cells, GS expression might acquire a ‘bad’ significance to the point that GS inhibition might be considered a conceivable strategy against cancer metastasis.

Phylogenomic analysis of secondary metabolism genes sheds light on their evolution in Aspergilli

DEFF Research Database (Denmark)

Theobald, Sebastian; Vesth, Tammi Camilla; Rasmussen, Jane Lind Nybo

.Natural products are encoded by genes located in close proximity, called secondary metabolic gene clusters, which makes them interesting targets for genomic analysis. We use a modified version of the Secondary Metabolite Unique Regions Finder (SMURF) algorithm, combined with InterPro annotations to create...... approximate maximum likelihood trees of conserved domains from secondary metabolic genes across 56 species, giving insights into the secondary metabolism gene diversity and evolution.In this study we can describe the evolution of non ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and hybrids.......In the aspMine project, we are sequencing and analyzing over 300 species of Aspergilli, agroup of filamentous fungi rich in natural compounds. The vast amount of data obtained from these species challenges the way we were mining for products and requires new pipelines for secondary metabolite analysis...

Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145

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Agnieszka Bera

2017-04-01

Full Text Available Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962, was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.

Identification of citrullinaemia carrier and detection of a new silent mutation at 240bp position in ASS1 gene of normal Holstein cattle

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Kotikalapudi Rosaiah

2014-01-01

Full Text Available The autosomal recessive genetic disorders are regularly investigated especially in Indian Holstein and Holstein Crossbred bulls before they entered in semen collection so that a defective gene should not be transmitted to future generations. Bovine citrullinaemia first reported in Australia is a metabolic disorder as one of the enzymes, Argininosuccinate synthetase (ASS involved in urea cycle is impaired in function. The mutation responsible for citrullinemia has been characterized as a single-base substitution at 256bp (C>T in coding exon 3 of argininosuccinate synthetase 1 (ASS1 gene, which converts the CGA (arginine at 86 codon to TGA (stop codon. A Holstein bull during routine molecular screening was found to be carrier for Citrullinaemia that was confirmed by sequencing. This is a fresh case of Citrullinaemia carrier in addition to three cases reported earlier in India. Partial sequencing of coding exon 3 of a normal Holstein revealed a new silent polymorphism at 240bp position that does not change amino acid (Sarine AGC>AGT at 80 codon within exon 3 of ASS1 gene. The sequence of exon 3 of ASS1 gene in a normal Holstein exhibiting a new polymorphism was submitted to NCBI with accession No. KF933365. The presence of citrullinaemia carriers in Indian Holstein, though in very low frequency, emphasizing to continue the investigation of mutant gene in cattle population.

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

Energy Technology Data Exchange (ETDEWEB)

Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation); Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S., E-mail: esipov@mx.ibch.ru [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation)

2017-01-15

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°Ð¡, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

International Nuclear Information System (INIS)

Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria

2006-01-01

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl 2 , as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

In silico identification and analysis of phytoene synthase genes in plants.

Science.gov (United States)

Han, Y; Zheng, Q S; Wei, Y P; Chen, J; Liu, R; Wan, H J

2015-08-14

In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

11th IUBMB Focused Meeting on the Aminoacyl-tRNA Synthetases: Sailing a New Sea of Complex Functions in Human Biology and Disease.

Science.gov (United States)

Francklyn, Christopher; Roy, Herve; Alexander, Rebecca

2018-05-01

The 11th IUBMB Focused Meeting on Aminoacyl-tRNA Synthetases was held in Clearwater Beach, Florida from 29 October⁻2 November 2017, with the aim of presenting the latest research on these enzymes and promoting interchange among aminoacyl-tRNA synthetase (ARS) researchers. Topics covered in the meeting included many areas of investigation, including ARS evolution, mechanism, editing functions, biology in prokaryotic and eukaryotic cells and their organelles, their roles in human diseases, and their application to problems in emerging areas of synthetic biology. In this report, we provide a summary of the major themes of the meeting, citing contributions from the oral presentations in the meeting.

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

Science.gov (United States)

Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Essential Non-Translational Functions of tRNA Synthetases

Science.gov (United States)

Guo, Min; Schimmel, Paul

2013-01-01

Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. While these new functions were thought to be ‘moonlighting activities’, many are as critical for cellular homeostasis as the activity in translation. New roles have been associated with cytoplasmic forms as well as with nuclear and secreted extracellular forms that impact pathways for cardiovascular development, the immune response, and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. Novel architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. While a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid binding site for another purpose. PMID:23416400

Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7T during Degradation of 3,3′-Dithiodipropionic Acid ▿ †

Science.gov (United States)

Schürmann, Marc; Wübbeler, Jan Hendrik; Grote, Jessica; Steinbüchel, Alexander

2011-01-01

The sucCD gene of Advenella mimigardefordensis strain DPN7T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCDAm) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCDAm is Mg2+ or Mn2+ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (Vmax = 9.85 ± 0.14 μmol min−1 mg−1, Km = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (Vmax = 0.12 ± 0.01 μmol min−1 mg−1) and the affinity was 6-fold lower (Km = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCDAm is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP. PMID:21515777

Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

International Nuclear Information System (INIS)

Fujita, Akiko; Sato, Chihiro; Kitajima, Ken.

2007-01-01

The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

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Nkosi Thokozani C

2011-07-01

Full Text Available Abstract Background The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

Science.gov (United States)

Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2016-03-28

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

CSIR Research Space (South Africa)

Kenyon, CP

2011-07-01

Full Text Available Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being...

Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

DEFF Research Database (Denmark)

Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

1990-01-01

enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5-phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor....... ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented....

Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.

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Eriko Kage-Nakadai

Full Text Available In multicellular organisms, the surface barrier is essential for maintaining the internal environment. In mammals, the barrier is the stratum corneum. Fatty acid transport protein 4 (FATP4 is a key factor involved in forming the stratum corneum barrier. Mice lacking Fatp4 display early neonatal lethality with features such as tight, thick, and shiny skin, and a defective skin barrier. These symptoms are strikingly similar to those of a human skin disease called restrictive dermopathy. FATP4 is a member of the FATP family that possesses acyl-CoA synthetase activity for very long chain fatty acids. How Fatp4 contributes to skin barrier function, however, remains to be elucidated. In the present study, we characterized two Caenorhabditis elegans genes, acs-20 and acs-22, that are homologous to mammalian FATPs. Animals with mutant acs-20 exhibited defects in the cuticle barrier, which normally prevents the penetration of small molecules. acs-20 mutant animals also exhibited abnormalities in the cuticle structure, but not in epidermal cell fate or cell integrity. The acs-22 mutants rarely showed a barrier defect, whereas acs-20;acs-22 double mutants had severely disrupted barrier function. Moreover, the barrier defects of acs-20 and acs-20;acs-22 mutants were rescued by acs-20, acs-22, or human Fatp4 transgenes. We further demonstrated that the incorporation of exogenous very long chain fatty acids into sphingomyelin was reduced in acs-20 and acs-22 mutants. These findings indicate that C. elegans Fatp4 homologue(s have a crucial role in the surface barrier function and this model might be useful for studying the fundamental molecular mechanisms underlying human skin barrier and relevant diseases.

The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

Science.gov (United States)

Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

2012-01-01

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

Identification of the Biosynthetic Gene Clusters for the Lipopeptides Fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum

DEFF Research Database (Denmark)

Sørensen, Jens Laurids; Sondergaard, Teis Esben; Covarelli, Lorenzo

2014-01-01

The closely related species Fusarium graminearum and Fusarium pseudograminearum differ in that each contains a gene cluster with a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) that is not present in the other species. To identify their products, we deleted PKS6 and NRPS7...... Fusarium species. On the basis of genes in the putative gene clusters we propose a model for biosynthesis where the polyketide product is shuttled to the NPRS via a CoA ligase and a thioesterase in F. pseudograminearum. In F. graminearum the polyketide is proposed to be directly assimilated by the NRPS....

Mammalian folylpoly-γ-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

International Nuclear Information System (INIS)

Cichowicz, D.J.; Shane, B.

1987-01-01

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K + was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied

Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

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Stefanie Berger

2012-01-01

Full Text Available The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM=0.27±0.05 mM that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell.

Origins of domestication and polyploidy in oca (Oxalis Tuberosa: Oxalidaceae). 2. Chloroplast-expressed glutamine synthetase data.

Science.gov (United States)

Emshwiller, Eve; Doyle, Jeff J

2002-07-01

In continuing study of the origins of the octoploid tuber crop oca, Oxalis tuberosa Molina, we used phylogenetic analysis of DNA sequences of the chloroplast-active (nuclear encoded) isozyme of glutamine synthetase (ncpGS) from cultivated oca, its allies in the "Oxalis tuberosa alliance," and other Andean Oxalis. Multiple ncpGS sequences found within individuals of both the cultigen and a yet unnamed wild tuber-bearing taxon of Bolivia were separated by molecular cloning, but some cloned sequences appeared to be artifacts of polymerase chain reaction (PCR) recombination and/or Taq error. Nonetheless, three classes of nonrecombinant sequences each joined a different part of the O. tuberosa alliance clade on the ncpGS gene tree. Octoploid oca shares two sequence classes with the Bolivian tuber-bearing taxon (of unknown ploidy level). Fixed heterozygosity of these two sequence classes in all ocas sampled suggests that they represent homeologous loci and that oca is allopolyploid. A third sequence class, found in eight of nine oca plants sampled, might represent a third homeologous locus, suggesting that oca may be autoallopolyploid, and is shared with another wild tuber-bearing species, tetraploid O. picchensis of southern Peru. Thus, ncpGS data identify these two taxa as the best candidates as progenitors of cultivated oca.

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

OpenAIRE

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-01-01

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

Gene adaptation to extreme environments

International Nuclear Information System (INIS)

Marlaire, P.; Rodriguez, V.; Kerner, N.

2005-01-01

Full text: This work is oriented to the study of gene adaptation to extreme conditions, such as the hydrothermal system located in Copahue, Neuquen, Argentina. The organisms living there develop under two pressure selection conditions: the high temperature of thermal water and the strong impact of ultraviolet (UV) radiation. Several microorganisms found in this region were isolated and different colonies resistant to UV radiation were selected, a Geobacillus thermoleovorans strain identified through 16S RNA sequence, being the most remarkable. A gene library was prepared out of this strain with UV sensitive bacteria BH200 (uvrA::Tn10). A number of clones were isolated by means of UV selection, the most outstanding being a gene carrier able to codify for the guanosine monophosphate synthetase enzyme (GMPs). The suitability of said enzyme was proved by means of additional assays performed on ght 1 bacteria (guaA26::Tn 10) which lacked the enzyme. A transcript of 1100 pb was detected through Northern Blot. The result was consistent with that obtained for the mapping of the starting transcription site. The cloned GMPs produces an increase in growth speed and a greater biomass in BH200 bacteria. (author)

Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: Mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing

OpenAIRE

Suo, Zucai; Chen, Huawei; Walsh, Christopher T.

2000-01-01

Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain [7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1 in YbtE] enzyme producing the virulence-conferring siderophore yersiniabactin in Yersinia pestis. The 350-kDa HMWP1 subunit contains a polyketide synthase module (KS-AT-MT2-KR-ACP) and a nonribosomal peptide synthetase module (Cy3-MT3-PCP3-TE). The full-length HMWP1 was heterologously overexpressed in Escherichia coli and purified...

Effect of intramolecular photochemical cross-linking and of alkylation of 4-thiouridine in E. coli tRNAsub(l)sup(Val). On the heterologous misccharging by yeast phenylalanyl-tRNA synthetase

Energy Technology Data Exchange (ETDEWEB)

Kumar, S A; Krauskopf, M; Ofengand, J [Roche Inst. of Molecular Biology, Nutley, N.J. (USA)

1973-08-01

The ability of yeast phenylalanyl-tRNA synthetase to carry out the heterologous mischarging of nine E. coli tRNAs with phenylalanine, and the presence of a common sequence in these tRNAs in the double stranded region adjacent to the dihydrouridine loop, have led to the proposal (by Dudock) that this region of the tRNA is involved in recognition by the yeast enzyme. The validity of this hypothesis has now been examined by chemical modification of the region in question using as a test tRNA, E. coli tRNA/sub 1/sup( val). Photochemical cross-linking of /sup 4/S(8) and C(13) by irradiation at 335 nm led to a complete loss of the ability of yeast phenylalanyl-tRNA synthetase to functionally recognize tRNA/sub 1/sup( val) and the rate of cross-linking was correlated with the rate of loss of activity when appropriate assay conditions were used. Cross-linking had no effect on the recognition by the homologous E. coli valyl-tRNA synthetase (EC 6.1.1.9). Similarly, S-alkylation of the /sup 4/S(8) residue with iodoacetamide at pH 9 yielded the uridine-4-thio(2-acetamide) derivative of tRNA with no loss of homologous recognition but with complete loss of heterologous charging activity. These results provide evidence that at least part of the yeast phenylalanyl-tRNA synthetase recognition site is located in the region of the tRNA proposed by Dudock, and, as a corollary, show that the E. coli valyl-tRNA synthetase recognition site(s) must be elsewhere in the molecule.

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

Science.gov (United States)

Haupt, V. Joachim; Schroeder, Michael; Labudde, Dirk

2018-01-01

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes. PMID:29659563

Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus

DEFF Research Database (Denmark)

O'Hanlon, Karen A.; Cairns, Timothy; Stack, Deirdre

2011-01-01

metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus....... Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase....

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

LENUS (Irish Health Repository)

Foy, Niall

2010-07-22

Abstract Background Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. Results A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. Conclusions The T box element controlling lysK (encoding LysRS1) expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.

Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis.

Science.gov (United States)

Jain, Vitul; Yogavel, Manickam; Oshima, Yoshiteru; Kikuchi, Haruhisa; Touquet, Bastien; Hakimi, Mohamed-Ali; Sharma, Amit

2015-05-05

The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ammonia stress on nitrogen metabolism in tolerant aquatic plant-Myriophyllum aquaticum.

Science.gov (United States)

Zhou, Qingyang; Gao, Jingqing; Zhang, Ruimin; Zhang, Ruiqin

2017-09-01

Ammonia has been a major reason of macrophyte decline in the water environment, and ammonium ion toxicity should be seen as universal, even in species frequently labeled as "NH 4 + specialists". To study the effects of high NH 4 + -N stress of ammonium ion nitrogen on tolerant submerged macrophytes and investigate the pathways of nitrogen assimilation in different organisms, Myriophyllum aquaticum was selected and treated with various concentrations of ammonium ions at different times. Increasing of ammonium concentration leads to an overall increase in incipient ammonia content in leaves and stems of plants. In middle and later stages, high concentrations of NH 4 + ion nitrogen taken up by M. aquaticum decreased, whereas the content of NO 3 - ion nitrogen increased. Moreover, in M. aquaticum, the activities of the enzymes nitrate reductase, glutamine synthetase and asparagine synthetase changed remarkably in the process of alleviating NH 4 + toxicity and deficiency. The results of the present study may support the studies on detoxification of high ammonium ion content in NH 4 + -tolerant submerged macrophytes and exploration of tissue-specific expression systems. Copyright © 2017. Published by Elsevier Inc.

The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

Science.gov (United States)

Middelkoop, T B; Labadie, R P

1985-01-01

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

In situ autoradiographic detection of folylpolyglutamate synthetase activity

International Nuclear Information System (INIS)

Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

1986-01-01

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6- 3 H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6- 3 H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells

Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases

Directory of Open Access Journals (Sweden)

Tukalo M. A.

2013-07-01

Full Text Available In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA rather than usual 4 or 5 nucleotides (type 1 tRNA. In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong

Overexpression of S-adenosyl-L-methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism.

Science.gov (United States)

Gong, Biao; Li, Xiu; VandenLangenberg, Kyle M; Wen, Dan; Sun, Shasha; Wei, Min; Li, Yan; Yang, Fengjuan; Shi, Qinghua; Wang, Xiufeng

2014-08-01

S-adenosyl-L-methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1-overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na(+) absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

A dual approach to study the electro-optical properties of a noncentrosymmetric L-asparagine monohydrate.

Science.gov (United States)

Shkir, Mohd; Muhammad, Shabbir; AlFaify, S; Irfan, Ahmad; Yahia, I S

2015-02-25

In this work we reports the experimental and theoretical investigation on an organic noncentrosymmetric monohydrated L-asparagine (LAM) molecule. LAM single crystals were grown in specially designed beaker for the first time. Structural confirmation was done by identifying the vibrational modes using IR and FT-Raman spectroscopic studies. The ultra violet-visible-near infrared absorbance, diffuse reflectance spectra were recorded in the spectral range 190-2500 nm. The optical transparency was calculated and found to be ∼80%. Its optical band gap was calculated found to be ∼5.100 eV. Density functional theory (DFT) was employed to optimize the molecular geometry of LAM using B3LYP/6-31G(∗) basis set of theory. The HOMO-LUMO energy gap of 6.047 eV and transition energy of 176 nm (f0=0.024) have been found in semi-quantitative agreement with our experimental results. The dipole moment, polarizability and first hyperpolarizability were calculated at the same level of theory. The obtained results reveals that the titled compound can be a decent contender for nonlinear applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome Sequence of Dickeya solani, a New soft Rot Pathogen of Potato, Suggests its Emergence May Be Related to a Novel Combination of Non-Ribosomal Peptide/Polyketide Synthetase Clusters

Directory of Open Access Journals (Sweden)

Linda Garlant

2013-12-01

Full Text Available Soft rot Enterobacteria in the genera Pectobacterium and Dickeya cause rotting of many crop plants. A new Dickeya isolate has been suggested to form a separate species, given the name Dickeya solani. This bacterium is spreading fast and replacing the closely related, but less virulent, potato pathogens. The genome of D. solani isolate D s0432-1 shows highest similarity at the nucleotide level and in synteny to D. dadantii strain 3937, but it also contains three large polyketide/fatty acid/non-ribosomal peptide synthetase clusters that are not present in D. dadantii 3937. These gene clusters may be involved in the production of toxic secondary metabolites, such as oocydin and zeamine. Furthermore, the D. solani genome harbors several specific genes that are not present in other Dickeya and Pectobacterium species and that may confer advantages for adaptation to new environments. In conclusion, the fast spreading of D. solani may be related to the acquisition of new properties that affect its interaction with plants and other microbes in the potato ecosystem.

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

Science.gov (United States)

Iqbal, Emil S; Dods, Kara K; Hartman, Matthew C T

2018-02-14

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic β-amino acids.

Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

International Nuclear Information System (INIS)

Duquerroy, Stephane; Vigouroux, Armelle; Rottier, Peter J.M.; Rey, Felix A.; Jan Bosch, Berend

2005-01-01

The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 A) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding 'zippers' that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle*

Science.gov (United States)

Dias, José; Renault, Louis; Pérez, Javier; Mirande, Marc

2013-01-01

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex. PMID:23836901

Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase

Science.gov (United States)

Martinis, S. A.; Fox, G. E.

1997-01-01

Recombinant E. coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine. [32P]-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging. Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent. The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively. Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

Science.gov (United States)

Esipov, R S; Abramchik, Yu A; Fateev, I V; Konstantinova, I D; Kostromina, M A; Muravyova, T I; Artemova, K G; Miroshnikov, A I

2016-01-01

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D -pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp . 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D -pentoses into 5-phosphates that were further converted into 5-phospho-α- D -pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D -ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

Science.gov (United States)

Gebril, Sayed; Seger, Mark; Villanueva, Fabiola Muro; Ortega, Jose Luis; Bagga, Suman; Sengupta-Gopalan, Champa

2015-10-01

Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Sherry L. Mowbray

2014-08-01

Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells.

Science.gov (United States)

Jastreboff, M M; Kedzierska, B; Rode, W

1983-07-15

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.

A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

OpenAIRE

Arriarán, Sofía; Agnelli, Silvia; Fernández López, José Antonio; Remesar Betlloch, Xavier; Alemany, Marià, 1946-

2012-01-01

A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium w...

De novo design and engineering of non-ribosomal peptide synthetases

Science.gov (United States)

Bozhüyük, Kenan A. J.; Fleischhacker, Florian; Linck, Annabell; Wesche, Frank; Tietze, Andreas; Niesert, Claus-Peter; Bode, Helge B.

2018-03-01

Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.

The Kallikrein-Kinin System in Bartter's Syndrome and Its Response to Prostaglandin Synthetase Inhibition

Science.gov (United States)

Vinci, Joseph M.; Gill, John R.; Bowden, Robert E.; Pisano, John J.; Izzo, Joseph L.; Radfar, Nazam; Taylor, Addison A.; Zusman, Randall M.; Bartter, Frederic C.; Keiser, Harry R.

1978-01-01

The kallikrein-kinin system was characterized in seven patients with Bartter's syndrome on constant metabolic regimens before, during, and after treatment with prostaglandin synthetase inhibitors. Patients with Bartter's syndrome had high values for plasma bradykinin, plasma renin activity (PRA), urinary kallikrein, urinary immunoreactive prostaglandin E excretion, and urinary aldosterone; urinary kinins were subnormal and plasma prekallikrein was normal. Treatment with indomethacin or ibuprofen which decreased urinary immunoreactive prostaglandin E excretion by 67%, decreased mean PRA (patients recumbent) from 17.3±5.3 (S.E.M.) ng/ml per h to 3.3±1.1 ng/ml per h, mean plasma bradykinin (patients recumbent) from 15.4±4.4 ng/ml to 3.9±0.9 ng/ml, mean urinary kallikrein excretion from 24.8±3.2 tosyl-arginine-methyl ester units (TU)/day to 12.4±2.0 TU/day, but increased mean urinary kinin excretion from 3.8±1.3 μg/day to 8.5±2.5 μg/day. Plasma prekallikrein remained unchanged at 1.4 TU/ml. Thus, with prostaglandin synthetase inhibition, values for urinary kallikrein and kinin and plasma bradykinin returned to normal pari passu with changes in PRA, in aldosterone, and in prostaglandin E. The results suggest that, in Bartter's syndrome, prostaglandins mediate the low urinary kinins and the high plasma bradykinin, and that urinary kallikrein, which is aldosterone dependent, does not control kinin excretion. The high plasma bradykinin may be a cause of the pressor hyporesponsiveness to angiotensin II which characterizes the syndrome. PMID:96139

Nitrile-synthesizing enzyme: Gene cloning, overexpression and application for the production of useful compounds.

Science.gov (United States)

Kumano, Takuto; Takizawa, Yuko; Shimizu, Sakayu; Kobayashi, Michihiko

2016-09-12

One of the nitrile-synthesizing enzymes, β-cyano-L-alanine synthase, catalyzes β-cyano-L-alanine (β-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the β-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of β-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.

Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii

International Nuclear Information System (INIS)

Fukunaga, Ryuya; Ishitani, Ryuichiro; Nureki, Osamu; Yokoyama, Shigeyuki

2004-01-01

The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. All five tRNA Leu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNA Leu , suggesting that they are LeuRS–tRNA Leu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNA Leu , with a corresponding crystal volume per protein weight of 2.9 Å 3 Da −1 and a solvent content of 57.3%

Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics.

Science.gov (United States)

Shaheen, Mohamed; Li, Jingru; Ross, Avena C; Vederas, John C; Jensen, Susan E

2011-12-23

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues. Copyright © 2011 Elsevier Ltd. All rights reserved.

Oligoadenylate synthetase 1 (OAS1 expression in human breast and prostate cancer cases, and its regulation by sex steroid hormones

Directory of Open Access Journals (Sweden)

Cláudio Jorge Maia

2016-06-01

Full Text Available Oligoadenylate synthetase 1 (OAS1 is an interferon-induced protein characterised by its capacity to catalyse the synthesis of 2ʹ-5ʹ-linked oligomers of adenosine from adenosine triphosphate (2-5A. The 2-5A binds to a latent Ribonuclease L (RNase L, which subsequently dimerises into its active form and may play an important role in the control of cell growth, differentiation and apoptosis. Previously, our research group identified OAS1 as a differentially-expressed gene in breast and prostate cancer cell lines when compared to normal cells. This study evaluates: i the expression of OAS1 in human breast and prostate cancer specimens; and ii the effect of sex steroid hormones in regulating the expression of OAS1 in breast (MCF-7 and prostate (LNCaP cancer cell lines. The obtained results showed that OAS1 expression was down-regulated in human infiltrative ductal carcinoma of breast, adenocarcinoma of prostate, and benign prostate hyperplasia, both at mRNA and protein level. In addition, OAS1 expression was negatively correlated with the progression of breast and prostate cancer. With regards to the regulation of OAS1 gene, it was demonstrated that 17β-estradiol (E2 down-regulates OAS1 gene in MCF-7 cell lines, an effect that seems to be dependent on the activation of oestrogen receptor (ER. On the other hand, 5α-dihydrotestosterone (DHT treatment showed no effect on the expression of OAS1 in LNCaP cell lines. The lower levels of OAS1 in breast and prostate cancer cases indicated that the OAS1/RNaseL apoptotic pathway may be compromised in breast and prostate tumours. Moreover, the present findings suggested that this effect may be enhanced by oestrogen in ER-positive breast cancers.

Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

DEFF Research Database (Denmark)

Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

1989-01-01

Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine...... represented a primary stimulus in all subjects. First, basal 2',5'A activity increased severalfold in response to yellow fever vaccination. In IDDM subjects, this increase was significantly lower (P = .025). Second, the 2',5'A activity increased proportionately to the higher basal 2',5'A activity in IDDM...

Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

Science.gov (United States)

Päri, Mailis; Kuusksalu, Anne; Lopp, Annika; Kjaer, Karina Hansen; Justesen, Just; Kelve, Merike

2014-02-01

2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

NARCIS (Netherlands)

Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M

1996-01-01

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high

Solid-phase peptide synthesis of isotocin with amide of asparagine protected with 1-tetralinyl. Trifluoromethanesulphonic acid (tfmsa deprotection, cleavage and air oxidation of mercapto groups to disulphide

Directory of Open Access Journals (Sweden)

Amir O. Yusuf

2001-12-01

Full Text Available Isotocin, a nonapeptide amide, was synthesised on a benzhydryl-resin using the Boc-strategy. Benzyl group was used in the protection of the side-chains of tyrosine, serine and cysteine. Tetralinyl group was used to protect asparagine side-chain. TFMSA-TFA-thioanisole-1,2-ethanedithiol (2:20:2:1 v/v was used on the peptide-resin under different cleavage conditions to obtain isotocin in a one-pot reaction. The cleavage at 40 °C for two hours gave isotocin quantitatively. Isotocin could be isolated in 61% yield.

One-step simultaneous differential scanning calorimetry-FTIR microspectroscopy to quickly detect continuous pathways in the solid-state glucose/asparagine Maillard reaction.

Science.gov (United States)

Hwang, Deng-Fwu; Hsieh, Tzu-Feng; Lin, Shan-Yang

2013-01-01

The stepwise reaction pathway of the solid-state Maillard reaction between glucose (Glc) and asparagine (Asn) was investigated using simultaneous differential scanning calorimetry (DSC)-FTIR microspectroscopy. The color change and FTIR spectra of Glc-Asn physical mixtures (molar ratio = 1:1) preheated to different temperatures followed by cooling were also examined. The successive reaction products such as Schiff base intermediate, Amadori product, and decarboxylated Amadori product in the solid-state Glc-Asn Maillard reaction were first simultaneously evidenced by this unique DSC-FTIR microspectroscopy. The color changed from white to yellow-brown to dark brown, and appearance of new IR peaks confirmed the formation of Maillard reaction products. The present study clearly indicates that this unique DSC-FTIR technique not only accelerates but also detects precursors and products of the Maillard reaction in real time.

Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.

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Sebastián, María; Anoz-Carbonell, Ernesto; Gracia, Begoña; Cossio, Pilar; Aínsa, José Antonio; Lans, Isaías; Medina, Milagros

2018-12-01

The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.

Action of. gamma. -radiation on the pH- and heat-stability of phenylalanine-tRNA-synthetases of cotton seeds

Energy Technology Data Exchange (ETDEWEB)

Karakuziev, T U [AN Uzbekskoj SSR, Tashkent. Inst. Biokhimii

1977-01-01

Action of temperature and pH on the activity of phenyl-alanine-tRNA-synthetases has been studied in two-day-old cotton seedlings grown from irradiated (25 and 50 kR) seeds. High thermolability and increased pH sensitivity were exhibited by enzymes E/sub 1/ and, particularly, E/sub 2/ of irradiated organisms as compared to those of the controls.

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

Science.gov (United States)

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

Science.gov (United States)

Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

2016-03-09

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene

NARCIS (Netherlands)

L.M. 't Hart (Leen); H.A.P. Pols (Huib); T. Hansen (Torben); I. Rietveld (Ingrid); J.M. Dekker (Jacqueline); J.A. Maassen (Johannes); M.G.A.A.M. Nijpels (Giel); G.M.C. Janssen (George); P.P. Arp (Pascal); R.J. Heine (Robert); A.G. Uitterlinden (André); T. Jorgensen (Torben); C.M. van Duijn (Cornelia); K. Borch-Johnsen; O. Pedersen (Oluf)

2005-01-01

textabstractPreviously, we have shown that a mutation in the mitochondrial DNA-encoded tRNA(Leu(UUR)) gene is associated with type 2 diabetes. One of the consequences of this mutation is a reduced aminoacylation of tRNA(Leu(UUR)). In this study, we have examined whether variants in the leucyl tRNA

Inactivation of Escherichia coli phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP. Identification of active site lysines

DEFF Research Database (Denmark)

Hilden, Ida; Hove-Jensen, Bjarne; Harlow, Kenneth W.

1995-01-01

M. Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit. oATP served as a substrate in the presence of ribose-5...

Small-angle X-ray-scattering investigation and structural-model study of the fatty-acid synthetase from pig liver

International Nuclear Information System (INIS)

Folkhard, W.; Felser, B.; Pilz, I.; Kratky, O.; Dutler, H.; Vogel, H.

1977-01-01

The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetase from this and other sources. When comparing small-angle X-ray-scattering curves calculated with these models and curves obtained from small-angle X-ray-scattering experiments carried out with the pig-liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and the functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an asymmetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other. (orig.) [de

Exploring the evolutionary diversity and assembly modes of multi-aminoacyl-tRNA synthetase complexes: lessons from unicellular organisms.

Science.gov (United States)

Laporte, Daphné; Huot, Jonathan L; Bader, Gaétan; Enkler, Ludovic; Senger, Bruno; Becker, Hubert Dominique

2014-11-28

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and ancient enzymes, mostly known for their essential role in generating aminoacylated tRNAs. During the last two decades, many aaRSs have been found to perform additional and equally crucial tasks outside translation. In metazoans, aaRSs have been shown to assemble, together with non-enzymatic assembly proteins called aaRSs-interacting multifunctional proteins (AIMPs), into so-called multi-synthetase complexes (MSCs). Metazoan MSCs are dynamic particles able to specifically release some of their constituents in response to a given stimulus. Upon their release from MSCs, aaRSs can reach other subcellular compartments, where they often participate to cellular processes that do not exploit their primary function of synthesizing aminoacyl-tRNAs. The dynamics of MSCs and the expansion of the aaRSs functional repertoire are features that are so far thought to be restricted to higher and multicellular eukaryotes. However, much can be learnt about how MSCs are assembled and function from apparently 'simple' organisms. Here we provide an overview on the diversity of these MSCs, their composition, mode of assembly and the functions that their constituents, namely aaRSs and AIMPs, exert in unicellular organisms. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

On the turn-inducing properties of asparagine: the structuring role of the amide side chain, from isolated model peptides to crystallized proteins.

Science.gov (United States)

Habka, S; Sohn, W Y; Vaquero-Vara, V; Géléoc, M; Tardivel, B; Brenner, V; Gloaguen, E; Mons, M

2018-01-31

Asparagine (Asn) is a powerful turn-inducer residue, with a large propensity to occupy the second position in the central region of β-turns of proteins. The present work aims at investigating the role of a local anchoring between the Asn side chain and the main chain in this remarkable property. For this purpose, the H-bonding patterns of an asparagine residue in an isolated protein chain fragment forming a γ- or a β-turn have been determined using IR/UV double resonance gas phase spectroscopy on laser-desorbed, jet-cooled short models in conjunction with relevant quantum chemistry calculations. These gas phase data provide evidence for an original double anchoring linking the Asn primary amide side chain (SC), which adopts a gauche+ rotameric form, to its main chain (MC) local environment. From both IR spectroscopic evidence (H-bond induced red shifts) and quantum chemistry, Asn SC is found to behave as a stronger H-bond acceptor than donor, resulting in stronger MC→SC H-bonds than SC→MC ones. These gas phase structural data, relevant to a hydrophobic environment, have been used as a reference to assess the anchoring taking place in high resolution crystallized proteins of the Protein Data Bank. This approach reveals that, when the SC adopts a gauche+ orientation, the stronger MC→SC bonds are preserved in many cases whereas the SC→MC bonds are always disrupted, in qualitative agreement with the gas phase ranking of these interactions. Most interestingly, when Asn occupies the second position of central part of a β-turn (i.e., the very turn-inducer position), the MC→SC H-bonds are also disrupted and replaced by a water-mediated SC to MC anchoring. Owing to the specific features of the hydrated Asn side chain, we propose that it could be a turn precursor structure, able to facilitate turn formation in the early events of the folding process.

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

Science.gov (United States)

Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

2015-04-01

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

Science.gov (United States)

Gutiérrez, Rodrigo A; Stokes, Trevor L; Thum, Karen; Xu, Xiaodong; Obertello, Mariana; Katari, Manpreet S; Tanurdzic, Milos; Dean, Alexis; Nero, Damion C; McClung, C Robertson; Coruzzi, Gloria M

2008-03-25

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Identification of astrocytoma associated genes including cell surface markers

International Nuclear Information System (INIS)

Boon, Kathy; Edwards, Jennifer B; Eberhart, Charles G; Riggins, Gregory J

2004-01-01

Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [1]. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie [2], and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes

Expansion, retention and loss in the Acyl-CoA synthetase "Bubblegum" (Acsbg) gene family in vertebrate history.

Science.gov (United States)

Lopes-Marques, Mónica; Machado, André M; Ruivo, Raquel; Fonseca, Elza; Carvalho, Estela; Castro, L Filipe C

2018-07-20

Fatty acids (FAs) constitute a considerable fraction of all lipid molecules with a fundamental role in numerous physiological processes. In animals, the majority of complex lipid molecules are derived from the transformation of FAs through several biochemical pathways. Yet, for FAs to enroll in these pathways they require an activation step. FA activation is catalyzed by the rate limiting action of Acyl-CoA synthases. Several Acyl-CoA enzyme families have been previously described and classified according to the chain length of FAs they process. Here, we address the evolutionary history of the ACSBG gene family which activates, FAs with >16 carbons. Currently, two different ACSBG gene families, ACSBG1 and ACSBG2, are recognized in vertebrates. We provide evidence that a wider and unequal ACSBG gene repertoire is present in vertebrate lineages. We identify a novel ACSBG-like gene lineage which occurs specifically in amphibians, ray finned fishes, coelacanths and cartilaginous fishes named ACSBG3. Also, we show that the ACSBG2 gene lineage duplicated in the Theria ancestor. Our findings, thus offer a far richer understanding on FA activation in vertebrates and provide key insights into the relevance of comparative and functional analysis to perceive physiological differences, namely those related with lipid metabolic pathways. Copyright © 2018 Elsevier B.V. All rights reserved.

Speciation of binary complexes of Pb(II and Cd(II with L-asparagine in dimethyl sulfoxide - water mixtures

Directory of Open Access Journals (Sweden)

C. N. Rao

2016-02-01

Full Text Available Chemical speciation of L-Asparagine complexes of Pb(II and Cd(II in presence of (0-50% v/v dimethyl sulfoxide(DMSO-water mixtures has been studied potentiometrically at 303.0 K and at an ionic strength of 0.16 mol L-1. The models containing different number of species were refined by using the computer program MINIQUAD75. The number of species in the models is chosen based on exhaustive modeling. The predominant species formed are of the type ML2, ML2H, and ML2H2. The best fit chemical models were chosen based on statistical parameters. The convenience of the models is ascertained by studying the effect of errors in concentrations of ingredients. The trend in variation of stability constants with change in the composition of medium is explained on the basis of predominant electrostatic and non-electrostatic forces. Chemical speciation was discussed based on the distribution diagrams. DOI: http://dx.doi.org/10.4314/bcse.v30i1.6

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

Science.gov (United States)

McClure, B; Mou, B; Canevascini, S; Bernatzky, R

1999-11-09

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions.

Science.gov (United States)

Singh, Kamal Krishna; Ghosh, Shilpi

2013-02-01

KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions.

Klebsiella pneumoniae asparagine tDNAs are integration hotspots for different genomic islands encoding microcin E492 production determinants and other putative virulence factors present in hypervirulent strains

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Andrés Esteban Marcoleta

2016-06-01

Full Text Available Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492 and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized.In this work we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mytomicin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least 7 protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we

The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment.

Science.gov (United States)

Saint-Léger, Adélaïde; Sinadinos, Christopher; Ribas de Pouplana, Lluís

2016-04-02

Malaria remains a major global health problem. Parasite resistance to existing drugs makes development of new antimalarials an urgency. The protein synthesis machinery is an excellent target for the development of new anti-infectives, and aminoacyl-tRNA synthetases (aaRS) have been validated as antimalarial drug targets. However, avoiding the emergence of drug resistance and improving selectivity to target aaRS in apicomplexan parasites, such as Plasmodium falciparum, remain crucial challenges. Here we discuss such issues using examples of known inhibitors of P. falciparum aaRS, namely halofuginone, cladosporin and borrelidin (inhibitors of ProRS, LysRS and ThrRS, respectively). Encouraging recent results provide useful guidelines to facilitate the development of novel drug candidates which are more potent and selective against these essential enzymes.

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Science.gov (United States)

Fairley, D J; Wang, G; Rensing, C; Pepper, I L; Larkin, M J

2006-12-01

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS)

Science.gov (United States)

Badgett, Majors J.; Boyes, Barry; Orlando, Ron

2017-05-01

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.

[ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism].

Science.gov (United States)

Lemeshko, V V; Kaliman, P A; Belostotskaia, L I; Uchitel', A A

1982-04-01

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors.

Science.gov (United States)

Rode, W; Jastreboff, M M

1984-01-01

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA

International Nuclear Information System (INIS)

Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S.

1987-01-01

Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the α 2 β 2 enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[ 14 C]tRNA/sub ox//sup Phe/ covalent complex indicates that the large (α, M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The [ 14 C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the α subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases

Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

International Nuclear Information System (INIS)

Wang, Xiaoying; Akasaka, Ryogo; Takemoto, Chie; Morita, Satoshi; Yamaguchi, Machiko; Terada, Takaho; Shirozu, Mikako; Yokoyama, Shigeyuki; Chen, Shilin; Si, Shuyi; Xie, Yong

2011-01-01

A hyperthermophilic adenylosuccinate synthetase from P. horikoshii OT3, which is 90–120 amino acids shorter than those from the vast majority of organisms, was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution. Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3 2 12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å 3 Da −1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution

Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans

Science.gov (United States)

McGill, Mitchell R.; Cao, Mengde; Svetlov, Archie; Sharpe, Matthew R.; Williams, C. David; Curry, Steven C.; Farhood, Anwar; Jaeschke, Hartmut; Svetlov, Stanislav I.

2014-01-01

Context New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. Objective Characterize ASS in APAP hepatotoxicity. Methods ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. Results In mice, ASS increased before injury, peaked before ALT, and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. Conclusions : ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity. PMID:24597531

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus.

Science.gov (United States)

Lehmacher, A; Vogt, A B; Hensel, R

1990-10-15

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

Asparaginase treatment side-effects may be due to genes with homopolymeric Asn codons (Review-Hypothesis)

Science.gov (United States)

BANERJI, JULIAN

2015-01-01

The present treatment of childhood T-cell leukemias involves the systemic administration of prokary-otic L-asparaginase (ASNase), which depletes plasma Asparagine (Asn) and inhibits protein synthesis. The mechanism of therapeutic action of ASNase is poorly understood, as are the etiologies of the side-effects incurred by treatment. Protein expression from genes bearing Asn homopolymeric coding regions (N-hCR) may be particularly susceptible to Asn level fluctuation. In mammals, N-hCR are rare, short and conserved. In humans, misfunctions of genes encoding N-hCR are associated with a cluster of disorders that mimic ASNase therapy side-effects which include impaired glycemic control, dislipidemia, pancreatitis, compromised vascular integrity, and neurological dysfunction. This paper proposes that dysregulation of Asn homeostasis, potentially even by ASNase produced by the microbiome, may contribute to several clinically important syndromes by altering expression of N-hCR bearing genes. By altering amino acid abundance and modulating ribosome translocation rates at codon repeats, the microbiomic environment may contribute to genome decoding and to shaping the proteome. We suggest that impaired translation at poly Asn codons elevates diabetes risk and severity. PMID:26178806

Asparaginase treatment side-effects may be due to genes with homopolymeric Asn codons (Review-Hypothesis).

Science.gov (United States)

Banerji, Julian

2015-09-01

The present treatment of childhood T-cell leukemias involves the systemic administration of prokaryotic L-asparaginase (ASNase), which depletes plasma Asparagine (Asn) and inhibits protein synthesis. The mechanism of therapeutic action of ASNase is poorly understood, as are the etiologies of the side-effects incurred by treatment. Protein expression from genes bearing Asn homopolymeric coding regions (N-hCR) may be particularly susceptible to Asn level fluctuation. In mammals, N-hCR are rare, short and conserved. In humans, misfunctions of genes encoding N-hCR are associated with a cluster of disorders that mimic ASNase therapy side-effects which include impaired glycemic control, dislipidemia, pancreatitis, compromised vascular integrity, and neurological dysfunction. This paper proposes that dysregulation of Asn homeostasis, potentially even by ASNase produced by the microbiome, may contribute to several clinically important syndromes by altering expression of N-hCR bearing genes. By altering amino acid abundance and modulating ribosome translocation rates at codon repeats, the microbiomic environment may contribute to genome decoding and to shaping the proteome. We suggest that impaired translation at poly Asn codons elevates diabetes risk and severity.

Genetic variants in nuclear-encoded mitochondrial genes influence AIDS progression.

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Sher L Hendrickson

2010-09-01

Full Text Available The human mitochondrial genome includes only 13 coding genes while nuclear-encoded genes account for 99% of proteins responsible for mitochondrial morphology, redox regulation, and energetics. Mitochondrial pathogenesis occurs in HIV patients and genetically, mitochondrial DNA haplogroups with presumed functional differences have been associated with differential AIDS progression.Here we explore whether single nucleotide polymorphisms (SNPs within 904 of the estimated 1,500 genes that specify nuclear-encoded mitochondrial proteins (NEMPs influence AIDS progression among HIV-1 infected patients. We examined NEMPs for association with the rate of AIDS progression using genotypes generated by an Affymetrix 6.0 genotyping array of 1,455 European American patients from five US AIDS cohorts. Successfully genotyped SNPs gave 50% or better haplotype coverage for 679 of known NEMP genes. With a Bonferroni adjustment for the number of genes and tests examined, multiple SNPs within two NEMP genes showed significant association with AIDS progression: acyl-CoA synthetase medium-chain family member 4 (ACSM4 on chromosome 12 and peroxisomal D3,D2-enoyl-CoA isomerase (PECI on chromosome 6.Our previous studies on mitochondrial DNA showed that European haplogroups with presumed functional differences were associated with AIDS progression and HAART mediated adverse events. The modest influences of nuclear-encoded mitochondrial genes found in the current study add support to the idea that mitochondrial function plays a role in AIDS pathogenesis.

Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

DEFF Research Database (Denmark)

Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.

2006-01-01

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using......, our data demonstrate that alpha-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity, although mutant forms of alpha-synuclein fail to restore this activity....

Linkage mapping of the gene for Type III collagen (COL3A1) to human chromosome 2q using a VNTR polymorphism

Energy Technology Data Exchange (ETDEWEB)

Tiller, G.E.; Polumbo, P.A.; Summar, M.L. (Vanderbilt Univ. Medical Center, Nashville, TN (United States))

1994-03-15

The gene for the [alpha]1(III) chain of type III collagen, COL3A1, has been previously mapped to human chromosome 2q24.3-q31 by in situ hybridization. Physical mapping by pulsed-field gel electrophoresis has demonstrated that COL3A1 lies within 35 kb of COL5A2. The authors genotyped the CEPH families at the COL3A2 locus using a pentanucleotide repeat polymorphism within intron 25. They demonstrated significant linkage to 18 anonymous markers as well as the gene for carbamyl phosphate synthetase (CPSI), which had been previously mapped to this region. No recombination was seen between COL3A1 and COL5A2 (Z = 9.93 at [theta] = 0) or D2S24 (Z = 10.55 at [theta] = 0). The locus order is (D2S32-D2S138-D2S148)-(D2S24-COL5A2-COL3A1)-(D2S118-D2S161), with odds of 1:2300 for the next most likely order. These relationships are consistent with the physical mapping of COL3A1 to the distal portion of 2q and place it proximal to CPSI by means of multipoint analysis. These linkage relationships should prove useful in further studies of Ehlers-Danlos syndrome type IV and carbamyl phosphate synthetase I deficiency and provide an additional framework for localizing other genes in this region. 13 refs., 2 figs., 1 tab.

JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

International Nuclear Information System (INIS)

Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R.

2006-01-01

Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML

Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

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Mónica Serrano

2015-04-01

Full Text Available Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

Arabidopsis plastidial folylpolyglutamate synthetase is required for seed reserve accumulation and seedling establishment in darkness.

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Hongyan Meng

Full Text Available Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3 of the plastidial folylpolyglutamate synthetase gene (AtDFB was defective in seed reserves and skotomorphogenesis. Lower carbon (C and higher nitrogen (N content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3-. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3- conditions, and further enhanced under NO3- limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3- during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3- as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis.

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains.

Science.gov (United States)

Shattuck, Jenifer E; Waechter, Aubrey C; Ross, Eric D

2017-07-04

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN + ], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

Dynamic Network Reconstruction from Gene Expression Data Describing the Effect of LiCl Stimulation on Hepatocytes

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Zellmer Sebastian

2005-12-01

Full Text Available Wnt/β-catenin signalling plays an important role in zonation of liver parenchyma and in patterning of hepatocyte heterogeneity. A characteristic marker of this heterogeneity is glutamine synthetase, which is expressed only in a subset of pericentrally located hepatocytes. To investigate, whether and how the Wnt/β-catenin signalling pathway is involved a culture of hepatocytes was stimulated by LiCl. This resulted in an increase in the specific GS activity, indicating that the Wnt/β-catenin pathway may participate in regulating GS levels. Affymetrix GeneChip oligonucleotide arrays were used to monitor the gene expression changes during a period from 2 to 24 hours after stimulation by LiCl. Samples from a cultivation without stimulation were used as controls. Based on the gene expression profiles a hypothetic signal transduction network was constructed by a reverse engineering algorithm. The network robustness was tested and the most stable structure was identified.

Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

LENUS (Irish Health Repository)

Kell, M R

2012-02-03

BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis

OpenAIRE

Sharma, Arvind; Sharma, Manmohan; Yogavel, Manickam; Sharma, Amit

2016-01-01

Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms) and platyhelminths (also called flatworms). These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs). These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs) are vital enzymes that decode genetic information and enable protein translation. ...

Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide

Energy Technology Data Exchange (ETDEWEB)

Chung, Hsiu-Ying, E-mail: hychung@mail.fcu.edu.tw; Li, Cheng Che

2013-03-01

A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3NSS peptide solution, and finally immersed in artificial saliva for 24 h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3NSS peptide compared to samples without peptide treatment. - Highlights: Black-Right-Pointing-Pointer The degree of nanohardness recovery of enamel was 4 times greater with the aid of 3NSS peptide. Black-Right-Pointing-Pointer 3NSS peptide promoted the formation of hydroxyapatites with

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

Science.gov (United States)

Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

2016-01-01

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

Science.gov (United States)

Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

2014-01-01

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

Science.gov (United States)

Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

2015-01-01

Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport.

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Pricilla E Day

Full Text Available Maternal environment and lifestyle factors may modify placental function to match the mother's capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content on a selection of metabolic and amino acid transporter genes and their associations with fetal growth.RNA was extracted from 102 term Southampton Women's Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR.Increased placental LAT2 (p = 0.01, y+LAT2 (p = 0.03, aspartate aminotransferase 2 (p = 0.02 and decreased aspartate aminotransferase 1 (p = 0.04 mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01, ASCT1 (p = 0.03, mitochondrial branched chain aminotransferase (p = 0.02 and glutamine synthetase (p = 0.05 was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05 associated with higher maternal diet quality (prudent dietary pattern pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05 and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01 associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01. Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001.A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking

Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis.

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Arvind Sharma

2016-11-01

Full Text Available Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms and platyhelminths (also called flatworms. These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs. These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs are vital enzymes that decode genetic information and enable protein translation. The specific inhibition of pathogen aaRSs bores well for development of next generation anti-parasitics. Here, we have identified and annotated aaRSs and accessory proteins from Loa loa (nematode and Schistosoma mansoni (flatworm to provide a glimpse of these protein translation enzymes within these parasites. Using purified parasitic lysyl-tRNA synthetases (KRSs, we developed series of assays that address KRS enzymatic activity, oligomeric states, crystal structure and inhibition profiles. We show that L. loa and S. mansoni KRSs are potently inhibited by the fungal metabolite cladosporin. Our co-crystal structure of Loa loa KRS-cladosporin complex reveals key interacting residues and provides a platform for structure-based drug development. This work hence provides a new direction for both novel target discovery and inhibitor development against eukaryotic pathogens that include L. loa and S. mansoni.

Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis.

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Sharma, Arvind; Sharma, Manmohan; Yogavel, Manickam; Sharma, Amit

2016-11-01

Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms) and platyhelminths (also called flatworms). These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs). These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs) are vital enzymes that decode genetic information and enable protein translation. The specific inhibition of pathogen aaRSs bores well for development of next generation anti-parasitics. Here, we have identified and annotated aaRSs and accessory proteins from Loa loa (nematode) and Schistosoma mansoni (flatworm) to provide a glimpse of these protein translation enzymes within these parasites. Using purified parasitic lysyl-tRNA synthetases (KRSs), we developed series of assays that address KRS enzymatic activity, oligomeric states, crystal structure and inhibition profiles. We show that L. loa and S. mansoni KRSs are potently inhibited by the fungal metabolite cladosporin. Our co-crystal structure of Loa loa KRS-cladosporin complex reveals key interacting residues and provides a platform for structure-based drug development. This work hence provides a new direction for both novel target discovery and inhibitor development against eukaryotic pathogens that include L. loa and S. mansoni.

Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen.

Science.gov (United States)

Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

2015-12-22

Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the

Antibacterial Property of a Coral-Associated Bacterium Pseudoalteromonas luteoviolacea Against Shrimp Pathogenic Vibrio harveyi (In Vitro Study

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OCKY KARNA RADJASA

2005-06-01

Full Text Available A coral-associated bacterium was successfully screened for secondary metabolites production based on PCR amplification of the nonribosomal peptide synthetase gene and was identified as closely related to Pseudoalteromonas luteoviolacea based on its 16S rDNA.The bacterium was found to inhibit the growth of shrimp pathogenic bacterium tested, Vibrio harveyi. To characterize the inhibiting metabolite, a 279 bp long DNA fragment was obtained and the deduced amino acid sequence showed conserved signature regions for peptide synthetases and revealed a high similarity to NosD (40% identity, a multifunctional peptide synthetase from Nostoc sp. GSV224, and NdaB (44% identity, a peptide synthetase module of Nodularia spumigena.

Antibacterial Property of a Coral-Associated Bacterium Pseudoalteromonas luteoviolacea Against Shrimp Pathogenic Vibrio harveyi (In Vitro Study

Directory of Open Access Journals (Sweden)

OCKY KARNA RADJASA

2005-06-01

Full Text Available A coral-associated bacterium was successfully screened for secondary metabolites production based on PCR amplification of the nonribosomal peptide synthetase gene and was identified as closely related to Pseudoalteromonas luteoviolacea based on its 16S rDNA. The bacterium was found to inhibit the growth of shrimp pathogenic bacterium tested, Vibrio harveyi. To characterize the inhibiting metabolite, a 279 bp long DNA fragment was obtained and the deduced amino acid sequence showed conserved signature regions for peptide synthetases and revealed a high similarity to NosD (40% identity, a multifunctional peptide synthetase from Nostoc sp. GSV224, and NdaB (44% identity, a peptide synthetase module of Nodularia spumigena

Influence of initial L-asparagine and glycerol concentrations on the batch growth kinetics of Mycobacterium bovis BCG Influência das concentrações iniciais de asparagina e glicerol sobre a cinética de crescimento submerso de Mycobacterium bovis

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Maria Betania Batista Leal

2004-12-01

Full Text Available The influences of the L-asparagine and glycerol initial concentrations in Sauton medium on the productivities of biomass and colony forming units were studied. The submerged batch cultivations of Mycobacterium bovis were carried out in a 20 L bioreactor. The L-asparagine and glycerol initial concentrations of 4.54 g/L and 25 mL/L, respectively, corresponded to the best biomass productivity, namely 2.5 g/L.day. On the other hand, the concentrations of 2.27 g/L and 25 mL/L, respectively, led to the highest productivity in terms of colony forming units, namely 2.7·10(6 colonies/mg.day. In addition, by means of the relative consumption analysis of L-asparagine and glycerol (50 and 26% respectively, it was concluded that the concentrations of such components could be reduced, with respect to the original Sauton medium composition, aiming the obtainment of an optimal BCG vaccine production in the bioreactor.Estudou-se a influência das concentrações iniciais, no meio de Sauton, de asparagina e glicerol sobre as produtividades, expressas em unidades formadoras de colônias e biomassa microbiana, referentes aos cultivos submersos do Mycobacterium bovis, em biorreator de 20 mL. As concentrações iniciais de 2,27 e 25 mL/L de asparagina e glicerol, respectivamente, conduziram à maior produtividade, em unidades formadoras de colônias, a saber 2,7.10(6 colônias/mg.dia. Por outro lado, as concentrações de 4,54 e 25 mL/L dos mesmos componentes, corresponderam à melhor produtividade em biomassa, a saber: 2,5 g/dia. Através das análises dos consumos relativos de asparagina e glicerol (50 e 26% respectivamente, verificou-se também que as concentrações destes componentes podem ser reduzidas na composição original do meio de Sauton, com o objetivo de obter uma produção otimizada de vacina BCG em bioreator.

Antioxidant activity of the melanoidin fractions formed from DGlucose and D-Fructose with L-Asparagine in the Maillard reaction

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A.P Echavarría

2013-03-01

Full Text Available Melanoidins formed at the last stage of the Maillard reaction have been shown to possess certain functional properties, such as antioxidant activity. In order to gain more insight into these functional properties, soluble model systems melanoidins from L‑Asparagine with D‑glucose or D‑fructose fractionating by ultrafiltration were analyzed. The fractionating/concentration sequence of the melanoidin fraction (1-300 kDa enabled five fractions to be produced.Additionally, the absorption of melanoidins was measured at different wavelengths (280, 325, 405 and browning at 420 nm. The fractionation effect of melanoidin systems on the color intensity, UV-absorbance scan wavelengths (nm, CIE, L*, a*, b* parameters and antioxidant activity were measured. For this purpose, antioxidant activity was evaluated through the free radical scavenging activity, including 1,1-diphenyl-2-picryl-hydrazil (DPPH and 2,20-azinobis (3-ethylbenothiazoline-6-sulfonic acid, diammonium salt (ABTS. The results showed that the absorption of the melanoidins formed from Glucose/L-Asn was higher than for those derived from Fructose/L-Asn. On the other hand, their antioxidant power was lower than that for melanoidins formed from Fructose/L-Asn systems.

Transcriptional regulation of the Hansenula polymorpha GSH2 gene in the response to cadmium ion treatment

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O. V. Blazhenko

2014-02-01

Full Text Available In a previous study we cloned GSH2 gene, encoding γ-glutamylcysteine synthetase (γGCS in the yeast Hansenula рolymorpha. In this study an analysis of molecular organisation of the H. рolymorpha GSH2 gene promoter was conducted and the potential binding sites of Yap1, Skn7, Creb/Atf1, and Cbf1 transcription factors were detected. It was established that full regulation of GSH2 gene expression in the response to cadmium and oxidative stress requires the length of GSH2 promoter to be longer than 450 bp from the start of translation initiation. To study the transcriptional regulation of H. polymorpha GSH2 gene recombinant strain, harbouring­ a reporter system, in which 1.832 kb regulatory region of GSH2 gene was fused to structural and terminatory regions of alcohol oxidase gene, was constructed. It was shown that maximum increase in H. polymorpha GSH2 gene transcription by 33% occurs in the rich medium under four-hour incubation with 1 μM concentration of cadmium ions. In the minimal medium the GSH2 gene expression does not correlate with the increased total cellular glutathione levels under cadmium ion treatment. We assume that the increased content of total cellular glutathione under cadmium stress in the yeast H. polymorpha probably is not controlled on the level of GSH2 gene transcription.

Beauvericin synthetase contains a calmodulin binding motif in the entomopathogenic fungus Beauveria bassiana.

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Kim, Jiyoung; Sung, Gi-Ho

2018-03-19

Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca 2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca 2+ signals are partially decoded by Ca 2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca 2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca 2+ . In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421-2538) binds to CaM in a Ca 2+ -dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.

Non-ribosomal peptide synthetases

Indian Academy of Sciences (India)

2017-01-19

Jan 19, 2017 ... knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic ..... The predictive power of the bioinformatics sequence analy- .... identified these motifs manually, though NaPDoS, anti-.

Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

Energy Technology Data Exchange (ETDEWEB)

Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

2004-05-18

Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster.

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Kumar, Abhishek; Henrissat, Bernard; Arvas, Mikko; Syed, Muhammad Fahad; Thieme, Nils; Benz, J Philipp; Sørensen, Jens Laurids; Record, Eric; Pöggeler, Stefanie; Kempken, Frank

2015-01-01

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals

DEFF Research Database (Denmark)

Black, P N; Færgeman, Nils J.; DiRusso, C C

2000-01-01

). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase...

A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

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Jan Mareš

Full Text Available A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea. Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

Construction of an engineering strain which knocked out the gene of thioesterase for Streptomyces parvus HCCB10043 and the reach of metabolites

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YIN Fang

2013-08-01

Full Text Available The major metabolites of Streptomyces parvus HCCB10043 is lipopeptide compounds A21978C,its genome sequence includes the non ribosomal peptide synthetase(NRPS,polyketide synthases(PKS and hybrid NRPS-PKS multi-enzyme system gene clusters,they do have a their common feature in the metabolite biosynthetic cluster,which is called TE domain as well.Thioesterase can synthesized the synthesis of compounds of the chain termination,and with functions to release mature lipopeptide hydrolysis and cyclized peptide chain aliphatic linear.This study,we knockout the TE domain of a gene cluster,which guide the biosynthesis of bipyridine,to obtain engineered bacteria.The fermentation results demonstrates reduced yields for metabolites 2,2′-Bipyridine (2,2′-BP.

The pulmonary histopathology of anti-KS transfer RNA synthetase syndrome.

Science.gov (United States)

Schneider, Frank; Aggarwal, Rohit; Bi, David; Gibson, Kevin; Oddis, Chester; Yousem, Samuel A

2015-01-01

The clinical spectrum of the antisynthetase syndromes (AS) has been poorly defined, although some frequently present with pulmonary manifestations. The anti-KS anti-asparaginyl-transfer RNA synthetase syndrome is one in which pulmonary interstitial lung disease is almost always present and yet the histopathologic spectrum is not well described. To define the morphologic manifestations of pulmonary disease in those patients with anti-KS antiasparaginyl syndrome. We reviewed the connective tissue disorder registry of the University of Pittsburgh and identified those patients with anti-KS autoantibodies who presented with interstitial lung disease and had surgical lung biopsies. The 5 patients with anti-KS antisynthetase syndrome were usually women presenting with dyspnea and without myositis, but with mechanic's hands (60%) and Raynaud phenomenon (40%). They most often presented with a usual interstitial pneumonia pattern of fibrosis (80%), with the final patient displaying organizing pneumonia. Pulmonary interstitial lung disease is a common presentation in patients with the anti-KS-antisynthetase syndrome, who are often women with rather subtle or subclinical connective tissue disease, whereas the literature emphasizes the nonspecific interstitial pneumonia pattern often diagnosed clinically. Usual interstitial pneumonia and organizing pneumonia patterns of interstitial injury need to be added to this clinical differential diagnosis.

Thermo-Regulation of Genes Mediating Motility and Plant Interactions in Pseudomonas syringae

Science.gov (United States)

Hockett, Kevin L.; Burch, Adrien Y.; Lindow, Steven E.

2013-01-01

Pseudomonas syringae is an important phyllosphere colonist that utilizes flagellum-mediated motility both as a means to explore leaf surfaces, as well as to invade into leaf interiors, where it survives as a pathogen. We found that multiple forms of flagellum-mediated motility are thermo-suppressed, including swarming and swimming motility. Suppression of swarming motility occurs between 28° and 30°C, which coincides with the optimal growth temperature of P. syringae. Both fliC (encoding flagellin) and syfA (encoding a non-ribosomal peptide synthetase involved in syringafactin biosynthesis) were suppressed with increasing temperature. RNA-seq revealed 1440 genes of the P. syringae genome are temperature sensitive in expression. Genes involved in polysaccharide synthesis and regulation, phage and IS elements, type VI secretion, chemosensing and chemotaxis, translation, flagellar synthesis and motility, and phytotoxin synthesis and transport were generally repressed at 30°C, while genes involved in transcriptional regulation, quaternary ammonium compound metabolism and transport, chaperone/heat shock proteins, and hypothetical genes were generally induced at 30°C. Deletion of flgM, a key regulator in the transition from class III to class IV gene expression, led to elevated and constitutive expression of fliC regardless of temperature, but did not affect thermo-regulation of syfA. This work highlights the importance of temperature in the biology of P. syringae, as many genes encoding traits important for plant-microbe interactions were thermo-regulated. PMID:23527276

L-Asparaginase from Streptomyces griseus NIOT-VKMA29: optimization of process variables using factorial designs and molecular characterization of L-asparaginase gene

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Meena, Balakrishnan; Anburajan, Lawrance; Sathish, Thadikamala; Vijaya Raghavan, Rangamaran; Dharani, Gopal; Valsalan Vinithkumar, Nambali; Kirubagaran, Ramalingam

2015-07-01

Marine actinobacteria are known to be a rich source for novel metabolites with diverse biological activities. In this study, a potential extracellular L-asparaginase was characterised from the Streptomyces griseus NIOT-VKMA29. Box-Behnken based optimization was used to determine the culture medium components to enhance the L-asparaginase production. pH, starch, yeast extract and L-asparagine has a direct correlation for enzyme production with a maximum yield of 56.78 IU mL-1. A verification experiment was performed to validate the experiment and more than 99% validity was established. L-Asparaginase biosynthesis gene (ansA) from Streptomyces griseus NIOT-VKMA29 was heterologously expressed in Escherichia coli M15 and the enzyme production was increased threefold (123 IU mL-1) over the native strain. The ansA gene sequences reported in this study encloses several base substitutions with that of reported sequences in GenBank, resulting in altered amino acid sequences of the translated protein.

Genetic variations in insulin-like growth factor binding protein acid labile subunit gene associated with growth traits in beef cattle (Bos taurus) in China.

Science.gov (United States)

Liu, Yu; Duan, Xiaoyan; Liu, Xiaolin; Guo, Jiazhong; Wang, Hongliang; Li, Zhixiong; Yang, Jing

2014-05-01

The insulin-like growth factor binding protein acid labile subunit (IGFALS) gene encodes a serum protein that binds to IGFs and regulates growth, development, and other physiological processes. We have found that sequencing of the IGFALS gene in Chinese Qinchuan beef cattle (n=300) revealed four SNP loci in exon two of the gene (g1219: T>C, g1893: T>C, g2612: G>A, and g2696: A>G). The SNP g2696: A>G resulted in a change from asparagine to aspartic acid (p. N574D) in the leucine-rich repeat region in the carboxyl-terminal domain of IGFALS. Four SNPs were in low linkage disequilibrium, and 12 different haplotypes were identified in the population. Association analysis suggested that SNP g1219: T>C had a significant association with hip width (PG displayed a significant association with stature (Pgrowth traits of bovine, and may serve as a genetic marker for selection of beef cattle for growth traits, including stature. Copyright © 2014 Elsevier B.V. All rights reserved.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

Science.gov (United States)

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

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Daniel P. Miller

2017-08-01

Full Text Available Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq. Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

Science.gov (United States)

Miller, Daniel P.; Hutcherson, Justin A.; Wang, Yan; Nowakowska, Zuzanna M.; Potempa, Jan; Yoder-Himes, Deborah R.; Scott, David A.; Whiteley, Marvin; Lamont, Richard J.

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism. PMID:28900609

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

Science.gov (United States)

Miller, Daniel P; Hutcherson, Justin A; Wang, Yan; Nowakowska, Zuzanna M; Potempa, Jan; Yoder-Himes, Deborah R; Scott, David A; Whiteley, Marvin; Lamont, Richard J

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis , and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

KAUST Repository

Hohl, Adrian

2017-12-06

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

KAUST Repository

Hohl, Adrian; Karan, Ram; Akal, Anstassja; Renn, Dominik; Liu, Xuechao; Dharamarajnadar, Alaguraj; Ghoprade, Seema Arun; Groll, Michael; Rueping, Magnus; Eppinger, Jö rg

2017-01-01

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

Antioxidant activity of the melanoidin fractions formed from D-Glucose and D-Fructose with L-Asparagine in the Maillard reaction

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A.P Echavarría

2013-01-01

Full Text Available Melanoidins formed at the last stage of the Maillard reaction have been shown to possess certain functional properties, such as antioxidant activity. In order to gain more insight into these functional properties, soluble model systems melanoidins from L - Asparagine with D - glucose or D - fructose fractionating by ultrafiltration were analyzed. The fractionating/concentration sequence of the melanoidin fraction (1 - 300 kDa enabled five fractions to be produced. Additionally, the absorption of melanoidins was measured at different wavelengths (280, 325, 405 and browning at 420 nm. The fractionati on effect of melanoidin systems on the color intensity, UV - absorbance scan wavelengths (nm, CIE, L*, a*, b* parameters and antioxidant activity were measured. For this purpose, antioxidant activity was evaluated through the free radical scavenging activit y, including 1,1 - diphenyl - 2 - picryl - hydrazil (DPPH and 2,20 - azinobis (3 - ethylbenothiazoline - 6 - sulfonic acid, diammonium salt (ABTS. The results showed that the absorption of the melanoidins formed from Glucose/L - Asn was higher than for those derived from Fructose/L - Asn. On the other hand, their antioxidant power was lower than that for melanoidins formed from Fructose/L - Asn systems.

Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

Science.gov (United States)

Galloway, D C; Blake, D G; Shepherd, A G; McLellan, L I

1997-11-15

We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

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Dilla Mareistia Fassah

2018-04-01

Full Text Available Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10 and steers (n = 10 of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01 and protein levels (1.4 fold; p< 0.05 of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05. Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05 and lactate dehydrogenase B (2.2 fold; p<0.01 genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05 and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05 genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial (3.5 fold; p<0.01 and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06 genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular

Motif-independent prediction of a secondary metabolism gene cluster using comparative genomics: application to sequenced genomes of Aspergillus and ten other filamentous fungal species.

Science.gov (United States)

Takeda, Itaru; Umemura, Myco; Koike, Hideaki; Asai, Kiyoshi; Machida, Masayuki

2014-08-01

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Substrate specificity and catalysis by the editing active site of alanyl-tRNA synthetase from Escherichia coli†

Science.gov (United States)

Pasman, Zvi; Robey-Bond, Susan; Mirando, Adam C.; Smith, Gregory J.; Lague, Astrid; Francklyn, Christopher S.

2011-01-01

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free standing P. horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNAAla and Ala-tRNAAla as substrates, the deacylation activities of the wild type and five different E. coli AlaRS editing site substitution mutants were characterized. The wild type AlaRS editing domain deacylated Ser-tRNAAla with a kcat/KM of 6.6 × 105 M−1 s−1, equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation, but only 12.2-fold greater than the rate with Ala-tRNAAla. While the E664A and T567G substitutions only minimally decreased kcat/KM, Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in kcat/KM in the range of 6-, 7.3-, and 15-fold. C666A AlaRS was 1.4-fold more active on Ala-tRNAAla relative to Ser-tRNAAla, providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine mis-incorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNAAla. PMID:21241052

Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase

Science.gov (United States)

Feun, L G; Marini, A; Walker, G; Elgart, G; Moffat, F; Rodgers, S E; Wu, C J; You, M; Wangpaichitr, M; Kuo, M T; Sisson, W; Jungbluth, A A; Bomalaski, J; Savaraj, N

2012-01-01

Background: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I–II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. Methods: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. Results: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS−), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS− patients receiving at least four doses at 320 IU m−2 was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. Conclusion: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression. PMID:22472884

Effect of Polymorphisms at Codon 146 of the Goat PRNP Gene on Susceptibility to Challenge with Classical Scrapie by Different Routes.

Science.gov (United States)

Papasavva-Stylianou, Penelope; Simmons, Marion Mathieson; Ortiz-Pelaez, Angel; Windl, Otto; Spiropoulos, John; Georgiadou, Soteria

2017-11-15

This report presents the results of experimental challenges of goats with scrapie by both the intracerebral (i.c.) and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by i.c. challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than those of animals of all other genotypes (chi-square value, 10.8; P = 0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN animals) succumbed to oral challenge. The results also indicate that any cases of infection in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the rapid enzyme-linked immunosorbent assay (ELISA) was more variable and dependent on the polymorphism. Together with data from previous studies of goats exposed to infection in the field, these data support the previously reported observations that polymorphisms at this codon have a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions. IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms that are associated with susceptibility or resistance to scrapie. However, in contrast to the polymorphisms in sheep, they are more numerous in goats and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. An initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after i.c. inoculation. The presence of a D or S residue prolonged incubation periods significantly, and prions were detected

3-Methylglutaconic aciduria, a frequent but underrecognized finding in carbamoyl phosphate synthetase I deficiency.

Science.gov (United States)

Rokicki, Dariusz; Pajdowska, Magdalena; Trubicka, Joanna; Thong, Meow-Keong; Ciara, Elżbieta; Piekutowska-Abramczuk, Dorota; Pronicki, Maciej; Sikora, Roman; Haidar, Rijad; Ołtarzewski, Mariusz; Jabłońska, Ewa; Muthukumarasamy, Premala; Sthaneswar, Pavai; Gan, Chin-Seng; Krajewska-Walasek, Małgorzata; Carrozzo, Rosalba; Verrigni, Daniela; Semeraro, Michela; Rizzo, Cristiano; Taurisano, Roberta; Alhaddad, Bader; Kovacs-Nagy, Reka; Haack, Tobias B; Dionisi-Vici, Carlo; Pronicka, Ewa; Wortmann, Saskia B

2017-08-01

The urea cycle disorder carbamoyl phosphate synthetase I deficiency is an important differential diagnosis in the encephalopathic neonate. This intoxication type inborn error of metabolism often leads to neonatal death or severe and irreversible damage of the central nervous system, even despite appropriate treatment. Timely diagnosis is crucial, but can be difficult on routine metabolite level. Here, we report ten neonates from eight families (finally) diagnosed with CPS1 deficiency at three tertiary metabolic centres. In seven of them the laboratory findings were dominated by significantly elevated urinary 3-methylglutaconic acid levels which complicated the diagnostic process. Our findings are both important for the differential diagnosis of patients with urea cycle disorders and also broaden the differential diagnosis of hyperammonemia associated with 3-methylglutaconic aciduria, which was earlier only reported in TMEM70 and SERAC1 defect. Copyright © 2017 Elsevier B.V. All rights reserved.

Advances in animal cell recombinant protein production: GS-NS0 expression system.

Science.gov (United States)

Barnes, L M; Bentley, C M; Dickson, A J

2000-02-01

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

Differential gene expression in the liver of the African lungfish, Protopterus annectens, after 6 months of aestivation in air or 1 day of arousal from 6 months of aestivation.

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Kum C Hiong

Full Text Available The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of

An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

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Pileggi Marcos

2001-01-01

Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

Icariin Is A PPARα Activator Inducing Lipid Metabolic Gene Expression in Mice

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Yuan-Fu Lu

2014-11-01

Full Text Available Icariin is effective in the treatment of hyperlipidemia. To understand the effect of icariin on lipid metabolism, effects of icariin on PPARα and its target genes were investigated. Mice were treated orally with icariin at doses of 0, 100, 200, and 400 mg/kg, or clofibrate (500 mg/kg for five days. Liver total RNA was isolated and the expressions of PPARα and lipid metabolism genes were examined. PPARα and its marker genes Cyp4a10 and Cyp4a14 were induced 2-4 fold by icariin, and 4-8 fold by clofibrate. The fatty acid (FA binding and co-activator proteins Fabp1, Fabp4 and Acsl1 were increased 2-fold. The mRNAs of mitochondrial FA β-oxidation enzymes (Cpt1a, Acat1, Acad1 and Hmgcs2 were increased 2-3 fold. The mRNAs of proximal β-oxidation enzymes (Acox1, Ech1, and Ehhadh were also increased by icariin and clofibrate. The expression of mRNAs for sterol regulatory element-binding factor-1 (Srebf1 and FA synthetase (Fasn were unaltered by icariin. The lipid lysis genes Lipe and Pnpla2 were increased by icariin and clofibrate. These results indicate that icariin is a novel PPARα agonist, activates lipid metabolism gene expressions in liver, which could be a basis for its lipid-lowering effects and its beneficial effects against diabetes.

Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model: Mechanism for deficient glutamatergic transmission?

Czech Academy of Sciences Publication Activity Database

Olabarria, M.; Noristani, H. N.; Verkhratsky, Alexei; Rodríguez Arellano, Jose Julio

2011-01-01

Roč. 6, č. 1 (2011), s. 55-63 ISSN 1750-1326 R&D Projects: GA ČR GA309/09/1696; GA ČR(CZ) GAP304/11/0184; GA ČR GA305/08/1384; GA ČR GA309/08/1381 Institutional research plan: CEZ:AV0Z50390703 Keywords : astroglia * glutamine synthetase * Alzheimer ?'?s disease Subject RIV: FH - Neurology Impact factor: 4.278, year: 2011

Complete Genome Sequence of the Endophytic Biocontrol Strain Bacillus velezensis CC09

OpenAIRE

Cai, Xunchao; Kang, Xingxing; Xi, Huan; Liu, Changhong; Xue, Yarong

2016-01-01

Bacillus velezensis is a heterotypic synonym of B. methylotrophicus, B. amyloliquefaciens subsp. plantarum, and Bacillus oryzicola, and has been used to control plant fungal diseases. In order to fully understand the genetic basis of antimicrobial capacities, we did a complete genome sequencing of the endophytic B.?velezensis strain CC09. Genes tightly associated with biocontrol ability, including nonribosomal peptide synthetases, polyketide synthetases, iron acquisition, colonization, and vo...

Activity of enzymes that hydrolyze sucrose and raffinose in the first stages of germination of Lactuca sativa cv. Grand rapids. [Invertase, alpha-galactosidose, and sucrose synthetase were observed

Energy Technology Data Exchange (ETDEWEB)

Slabnik, E.; Calderon, P.; Diaz, H.

1981-01-01

The activities of enzymes capable of metabolizing raffinose and sucrose on achenes of lettuce were studied. During the first stages of germination, evidence was obtained for the occurrence of invertase in the endosperm and embryonic axis. Alpha-galactosidase was localized in the endosperm and cotyledons. Sucrose synthetase was present in the dry seed.

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

Science.gov (United States)

Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

2016-11-01

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera

DEFF Research Database (Denmark)

Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just

2009-01-01

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have...... not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2′-PDE activity, which highlights the probable existence of a premature 2–5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2–5A degrading activity....... Upon this finding, two out of three elements forming the 2–5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis...

2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

Science.gov (United States)

Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just; Kelve, Merike; Uriz, Maria Jesus

2009-01-01

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

Science.gov (United States)

Chen, Yaozong; Li, Tin Lok; Lin, Xingbang; Li, Xin; Li, Xiang David; Guo, Zhihong

2017-07-21

o -Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Initial organic products of fixation of [13N]dinitrogen by root nodules of soybean (Glycine max)

International Nuclear Information System (INIS)

Meeks, J.C.; Wolk, C.P.; Schilling, N.; Shaffer, P.W.; Avissar, Y.; Chien, W.S.

1978-01-01

When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate ( 13 N)N 2 , the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; 13 N in alanine becomes detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major 13 N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas ( 13 N)glutamine comprises only a small fraction of organic 13 N, and very little 13 N is detected in asparagine. The fixation of ( 13 N)N 2 into organic products was inhibited more than 99 percent by C 2 H 2 (10 percent, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules

Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

International Nuclear Information System (INIS)

Jensen, R.A.

1983-01-01

Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

Structural characterization of Helicobacter pylori dethiobiotin synthetase reveals differences between family members

Energy Technology Data Exchange (ETDEWEB)

Porebski, Przemyslaw J.; Klimecka, Maria; Chruszcz, Maksymilian; Nicholls, Robert A.; Murzyn, Krzysztof; Cuff, Marianne E.; Xu, Xiaohui; Cymborowski, Marcin; Murshudov, Garib N.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (MCSG); (UV); (MRC)

2012-07-11

Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS-ATP and hpDTBS-GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.

Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates

OpenAIRE

Rayevsky A. V.; Tukalo M. A.

2016-01-01

Aim. To investigate the structural bases for the amino acid selectivity of the Thermus thermophilus leucyl-tRNA synthetase (LeuRSTT) aminoacylation site and to disclose the binding pattern of pre-transfer editing substrates. Methods. Eight amino acids proposed as semi-cognate substrates for aminoacylation and eight aminoacyl-adenylates (formed from AMP and eight amino acids) were prepared in zwitterions form. The protein structure with a co-crystallized substrate in the aminoacylation site [P...

Differential neutrophil gene expression in early bovine pregnancy

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Kizaki Keiichiro

2013-02-01

Full Text Available Abstract Background In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT-stimulated gene expression in peripheral blood leukocytes (PBL, was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods PBL were collected on days 0 (just before artificial insemination, 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15, myxovirus-resistance (MX 1 and 2, and 2′-5′-oligoadenylate synthetase (OAS1, were then analyzed in each fraction through day 28 of gestation using qPCR. Results Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte

The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features

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Aklujkar, Muktak [University of Massachusetts, Amherst; Haveman, Shelley [University of Massachusetts, Amherst; DiDonatoJr, Raymond [University of Massachusetts, Amherst; Chertkov, Olga [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Brown, Peter [University of Massachusetts, Amherst; Lovley, Derek [University of Massachusetts, Amherst

2012-01-01

Background: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. Results: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate: ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. Conclusions: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

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da Silva Neto José F

2010-08-01

Full Text Available Abstract Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase, was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

Emiliania Huxleyi (Prymnesiophyceae): Nitrogen-metabolism genes and their expression in response to external nitrogen souces

DEFF Research Database (Denmark)

Bruhn, Annette; LaRoche, Julie; Richardson, Katherine

2010-01-01

The availability and composition of dissolved nitrogen in ocean waters are factors that influence species composition in natural phytoplankton communities. The same factors affect the ratio of organic to inorganic carbon incorporation in calcifying species, such as the coccolithophore Emiliania...... huxleyi (Lohman) W. W. Hay et H. Mohler. E. huxleyi has been shown to thrive on various nitrogen sources, including dissolved organic nitrogen. Nevertheless, assimilation of dissolved nitrogen under nitrogen-replete and -limited conditions is not well understood in this ecologically important species....... In this study, the complete amino acid sequences for three functional genes involved in nitrogen metabolism in E. huxleyi were identified: a putative formamidase, a glutamine synthetase (GSII family), and assimilatory nitrate reductase. Expression patterns of the three enzymes in cells grown on inorganic...

Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.

Science.gov (United States)

Zhou, Jie J; Wang, Feng; Xu, Zhiwen; Lo, Wing-Sze; Lau, Ching-Fun; Chiang, Kyle P; Nangle, Leslie A; Ashlock, Melissa A; Mendlein, John D; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2014-07-11

Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

DEFF Research Database (Denmark)

Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

2005-01-01

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine....... However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

Science.gov (United States)

Rubio, Manuel; Ballester, Ana Rosa; Olivares, Pedro Manuel; Castro de Moura, Manuel; Dicenta, Federico; Martínez-Gómez, Pedro

2015-01-01

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Structural Analysis of Xylanase from Marine Thermophilic Geobacillus stearothermophilus in Tanjung Api, Poso, Indonesia

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BUDI SAKSONO

2010-12-01

Full Text Available A xylanase gene, xynA, has been cloned from thermophilic strain Geobacillus stearothermophilus, which was isolated from marine Tanjung Api, Indonesia. The polymerase chain reaction product of 1266 bp of xynA gene consisted of 1221 bp open reading frame and encoded 407 amino acids including 30 residues of signal peptide. The sequence exhibited highest identity of 98.7% in the level of amino acid, with an extracellular endo-1,4-â-xylanase from G stearothermophilus T-6 (E-GSX T-6 of the glycoside hydrolase family 10 (GH10. A comparative study between the local strain G. stearothermophilus (GSX L and E-GSX T-6 on homology of amino acid sequence indicated five differents amino acids in the gene. They were Threonine/Alanine (T/A, Asparagine/Aspartate (N/D, Lysine/Asparagine (K/N, Isoleucine/Methionine (I/M, Serine/Threonine (S/T at the position 220, 227, 228, 233, and 245, respectively. Protein structural analysis of those differences suggested that those amino acids may play role in biochemical properties such as enzyme stability, in particular its thermostability.

Ammonium assimilation in rice based on the occurrence of 15N and inhibition of glutamine synthetase activity

Energy Technology Data Exchange (ETDEWEB)

Magalhaes, J. R.; Huber, D. M.; Lee, T. C.; Tsai, C. Y.

1995-07-01

Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two-week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX) an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N-labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gin) and glutamic acid (Glu) before being converted to other amino acids through transamination. The formation of amino acids such as aspartic acid (Asp) and alanine (Ala) directly from free NH4 in roots also has been reported. Translocation of free NH4 to plant shoots, based on the concentration of free NH4 in xylem exudate, has been reported in tomato, although NH4 in shoots primarily originates from nitrate reduction in the shoot. Photorespiration also can contribute to the accumulation of NH4 in leaves. The GS/GOGAT cycle appears to be primarily responsible for the assimilation of exogenously supplied NH4 and NH4 derived from nitrate reduction in leaves, as well as NH4 derived from photorespiration. Genetic evidence cited to support this conclusion includes the lethal effect of photorespiratory conditions on plant mutants deficient in chloroplast-localized GS and GOGAT activities, and the rapid accumulation of free NH4 in GS-deficient mutants under photorespiratory conditions. The present study was initiated to quantify the in vivo amino acid synthesis in rice

Ammonium assimilation in rice based on the occurrence of 15N and inhibition of glutamine synthetase activity

International Nuclear Information System (INIS)

Magalhaes, J.R.; Huber, D.M.; Lee, T.C.; Tsai, C.Y.

1995-01-01

Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two-week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX) an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N-labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gin) and glutamic acid (Glu) before being converted to other amino acids through transamination. The formation of amino acids such as aspartic acid (Asp) and alanine (Ala) directly from free NH4 in roots also has been reported. Translocation of free NH4 to plant shoots, based on the concentration of free NH4 in xylem exudate, has been reported in tomato, although NH4 in shoots primarily originates from nitrate reduction in the shoot. Photorespiration also can contribute to the accumulation of NH4 in leaves. The GS/GOGAT cycle appears to be primarily responsible for the assimilation of exogenously supplied NH4 and NH4 derived from nitrate reduction in leaves, as well as NH4 derived from photorespiration. Genetic evidence cited to support this conclusion includes the lethal effect of photorespiratory conditions on plant mutants deficient in chloroplast-localized GS and GOGAT activities, and the rapid accumulation of free NH4 in GS-deficient mutants under photorespiratory conditions. The present study was initiated to quantify the in vivo amino acid synthesis in rice

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

Science.gov (United States)

Amoutzias, Grigoris D.; Chaliotis, Anargyros; Mossialos, Dimitris

2016-01-01

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds. PMID:27092515

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

Directory of Open Access Journals (Sweden)

Grigoris D. Amoutzias

2016-04-01

Full Text Available Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs and polyketides (PKs are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes and type-I polyketide synthases (PKSes-I, respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

Energy Technology Data Exchange (ETDEWEB)

Cura, Vincent; Olieric, Natacha; Guichard, Alexandre [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Wang, En-Duo [State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031 (China); Moras, Dino [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Eriani, Gilbert [Architecture et Réactivité de l’ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 Rue René Descartes, 67084 Strasbourg (France); Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2005-10-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques.

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

International Nuclear Information System (INIS)

Cura, Vincent; Olieric, Natacha; Guichard, Alexandre; Wang, En-Duo; Moras, Dino; Eriani, Gilbert; Cavarelli, Jean

2005-01-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

Science.gov (United States)

Tamano, Koichi; Miura, Ai

2016-09-01

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole (Cynoglossus semilaevis.

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Hua Guo

Full Text Available Aquaporin 1 (AQP1 is a member of the transmembrane water channel family of proteins with special structural features, and two AQP1 paralogous genes (aqp1aa and aqp1ab are reported in teleosts. In the present study, the aqp1aa gene of half-smooth tongue sole (Cynoglossus semilaevis was cloned and characterized. The full-length cDNA of aqp1aa is 1411 bp with a 786 bp open reading frame encoding a 261-amino acid putative protein with a characteristic structure consisting of 6 membrane-spanning α-helical domains and two highly conserved asparagine-proline-alanine motifs. Real-time quantitative PCR revealed that aqp1aa mRNA is expressed predominantly in the testis of males and pseudo-males, while its expression is low in the ovary and lowest in doublesex and mab-3-related transcription factor 1(DMRT1 knock out fish and triploid males. In situ hybridization indicated that aqp1aa mRNA is expressed mainly in the germ cells of males and pseudo-males, especially in spermatozoa and spermatids. These results suggest that the aqp1aa may play a role in spermatogenesis of C. semilaevis.

Effect of 60Co γ radiation on the valyl-tRNA synthetase isolated from chick embryo brain

International Nuclear Information System (INIS)

Boloni, E.; Szabo, L.D.

1978-01-01

he effect of 60 Coγ irradiation on the activity of valyl-tRNA synthetase isolated from chick embryo brain was studied. The enzyme activity exponentially decreased in the dose range 10 to 200 krad. The first step of the enzyme action, i.e. amino acid activation, was found to be less sensitive to irradiation than the whole reaction, the formation of valyl-tRNA. 2-Mercapto ethanol and/or glycerol had a significant radioprotective effect. The lesion caused by radiation in the enzyme was also influenced by its concentration during exposure (diluted effect). According to gel-electrophoretic experiments, no chain rupture occurred in the enzyme molecule. Not even a change in Ksub(m) was observed; however, the maximum velocity of the reaction was found to decrease with increasing radiation dose. (author)

In silico analysis highlights the frequency and diversity of type 1 lantibiotic gene clusters in genome sequenced bacteria

LENUS (Irish Health Repository)

Marsh, Alan J

2010-11-30

Abstract Background Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria. Results In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production. Conclusions Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.

Draft genome sequence and chemical profiling of Fusarium langsethiae, an emerging producer of type A trichothecenes

DEFF Research Database (Denmark)

Lysøe, Erik; Frandsen, Rasmus John Normand; Divon, Hege H.

2016-01-01

. The assembly was fragmented, but reveals a genome of approximately 37.5 Mb, with a GC content around 48%, and 12,232 predicted protein-coding genes. Focusing on secondary metabolism we identified candidate genes for 12 polyketide synthases, 13 non-ribosomal peptide synthetases, and 22 genes for terpene/isoprenoid...

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

Science.gov (United States)

Krajewski, Wojciech W; Collins, Ruairi; Holmberg-Schiavone, Lovisa; Jones, T Alwyn; Karlberg, Tobias; Mowbray, Sherry L

2008-01-04

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening.

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Böttcher, Christine; Keyzers, Robert A; Boss, Paul K; Davies, Christopher

2010-08-01

In fleshy fruit, levels of indole-3-acetic acid (IAA), the most abundant auxin, decline towards the onset of ripening. The application of auxins to immature fruit can delay the ripening processes. However, the mechanisms by which the decrease in endogenous IAA concentrations and the maintenance of low auxin levels in maturing fruit are achieved remain elusive. The transcript of a GH3 gene (GH3-1), encoding for an IAA-amido synthetase which conjugates IAA to amino acids, was detected in grape berries (Vitis vinifera L.). GH3-1 expression increased at the onset of ripening (veraison), suggesting that it might be involved in the establishment and maintenance of low IAA concentrations in ripening berries. Furthermore, this grapevine GH3 gene, responded positively to the combined application of abscisic acid and sucrose and to ethylene, linking it to the control of ripening processes. Levels of IAA-aspartic acid (IAA-Asp), an in vitro product of recombinant GH3-1, rose after veraison and remained high during the following weeks of the ripening phase when levels of free IAA were low. A similar pattern of changes in free IAA and IAA-Asp levels was detected in developing tomatoes (Solanum lycopersicum Mill.), where low concentrations of IAA and an increase in IAA-Asp concentrations coincided with the onset of ripening in this climacteric fruit. Since IAA-Asp might be involved in IAA degradation, the GH3 catalysed formation of this conjugate at, and after, the onset of ripening could represent a common IAA inactivation mechanism in climacteric and non-climacteric fruit which enables ripening.

The nitrate-reduction gene cluster components exert lineage-dependent contributions to optimization of Sinorhizobium symbiosis with soybeans.

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Liu, Li Xue; Li, Qin Qin; Zhang, Yun Zeng; Hu, Yue; Jiao, Jian; Guo, Hui Juan; Zhang, Xing Xing; Zhang, Biliang; Chen, Wen Xin; Tian, Chang Fu

2017-12-01

Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix - ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix + ) but ineffective (Eff - ) nodules. These Fix + /Eff - nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype

International Nuclear Information System (INIS)

Kowara, Renata; Karaczyn, Aldona; Cheng, Robert Y.S.; Salnikow, Konstantin; Kasprzak, Kazimierz S.

2005-01-01

B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

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Araújo, Welington Luiz; Santos, Daiene Souza; Dini-Andreote, Francisco; Salgueiro-Londoño, Jennifer Katherine; Camargo-Neves, Aline Aparecida; Andreote, Fernando Dini; Dourado, Manuella Nóbrega

2015-10-01

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in

Facilitated stimulus-response associative learning and long-term memory in mice lacking the NTAN1 amidase of the N-end rule pathway.

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Balogh, S A; McDowell, C S; Tae Kwon, Y; Denenberg, V H

2001-02-23

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Inactivation of the NTAN1 gene encoding the asparagine-specific N-terminal amidase in mice results in impaired spatial memory [26]. The studies described here were designed to further characterize the effects upon learning and memory of inactivating the NTAN1 gene. NTAN1-deficient mice were found to be better than wild-type mice on black-white and horizontal-vertical discrimination learning. They were also better at 8-week Morris maze retention testing when a reversal trial was not included in the testing procedures. In all three tasks NTAN1-deficient mice appeared to use a strong win-stay strategy. It is concluded that inactivating the asparagine-specific branch of the N-end rule pathway in mice results in impaired spatial learning with concomitant compensatory restructuring of the nervous system in favor of non-spatial (stimulus-response) learning.

Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

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Samiha Sioud

2007-01-01

Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

[Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

Science.gov (United States)

Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

2018-01-01

Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

Gene Expression Analysis of Plum pox virus (Sharka Susceptibility/Resistance in Apricot (Prunus armeniaca L..

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Manuel Rubio

Full Text Available RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925, which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein PPVres region could also be involved in the resistance.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

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Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

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Tulika Munshi

Full Text Available ATP-dependent Mur ligases (Mur synthetases play essential roles in the biosynthesis of cell wall peptidoglycan (PG as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne

1989-01-01

A DNA fragment encoding kanamycin resistance was inserted in vitro into a plasmid-borne prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. The resulting plasmids were subsequently transferred to the chromosome by homologous recombination and the haploid strains prs-3::Kan......R and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show...

Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli

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Kormelink Tom

2012-05-01

Full Text Available Abstract Background The assimilation of nitrogen in bacteria is achieved through only a few metabolic conversions between alpha-ketoglutarate, glutamate and glutamine. The enzymes that catalyze these conversions are glutamine synthetase, glutaminase, glutamate dehydrogenase and glutamine alpha-ketoglutarate aminotransferase. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR. Results Although we observed a substantial difference in the extent of central nitrogen metabolism in the various species, the basic GlnR regulon was remarkably constant and appeared not affected by the presence or absence of the other three main regulators. We found a conserved regulatory association of GlnR with glutamine synthetase (glnRA operon, and the transport of ammonium (amtB-glnK and glutamine/glutamate (i.e. via glnQHMP, glnPHQ, gltT, alsT. In addition less-conserved associations were found with, for instance, glutamate dehydrogenase in Streptococcaceae, purine catabolism and the reduction of nitrite in Bacillaceae, and aspartate/asparagine deamination in Lactobacillaceae. Conclusions Our analyses imply GlnR-mediated regulation in constraining the import of ammonia/amino-containing compounds and the production of intracellular ammonia under conditions of high nitrogen availability. Such a role fits with the intrinsic need for tight control of ammonia levels to limit futile cycling.

Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.

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Cho Yeow Koh

2014-04-01

Full Text Available Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general 'ATP-engaging' binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.

The α1,6-fucosyltransferase gene (fut8 from the Sf9 lepidopteran insect cell line: insights into fut8 evolution.

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Sylvie Juliant

Full Text Available The core alpha1,6-fucosyltransferase (FUT8 catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked N-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the Sf9 (Spodoptera frugiperda lepidopteran cell line. As in most animal genomes, fut8 is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran fut8 genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the fut8 genes in metazoans. Analysis of the intron-exon organization in 56 fut8 orthologs allowed us to propose a model for fut8 evolution in metazoans. The presence of a highly variable number of exons in metazoan fut8 genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera fut8 genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.

Lincosamide Synthetase-A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

Czech Academy of Sciences Publication Activity Database

Janata, Jiří; Kadlčík, Stanislav; Koběrská, Markéta; Ulanová, Dana; Kameník, Zdeněk; Novák, Petr; Kopecký, Jan; Novotná, Jitka; Radojevič, Bojana; Plháčková, Kamila; Gažák, Radek; Najmanová, Lucie

2015-01-01

Roč. 10, č. 3 (2015) E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : BIOSYNTHETIC GENE-CLUSTER * MOLECULAR-WEIGHT THIOL * PHOSPHOPANTETHEINYL TRANSFERASE Subject RIV: EE - Microbiology, Virology Impact factor: 3.057, year: 2015

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

Science.gov (United States)

Lin, Li-Ching; Chen, Chi-Fen; Ho, Chun-Te; Liu, Jun-Jen; Liu, Tsan-Zon; Chern, Chi-Liang

2018-04-01

This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ‑glutamylcysteine synthetase heavy chain (γ‑GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ‑GCSh activity were measured by HPLC methods. γ‑GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ‑GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ‑GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. Our data unequivocally demonstrate that γ‑GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression▿†

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Pearson, Melanie M.; Yep, Alejandra; Smith, Sara N.; Mobley, Harry L. T.

2011-01-01

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract. PMID:21505083

Ability of secondary metabolites from trichoderma virens to mediate communication during mutualistic or pathogenic interactions

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A bioinformatic study was conducted to identify the putative genes in the biocontrol agent Trichoderma virens that encode for non-ribosomal peptide synthetases (NRPS). Gene expression analysis of 22 putative NRPSs and 4 NRPS/PKS (polyketide synthase) hybrid enzymes was conducted in the presence and...

Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

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Zhiliang Yu

2015-01-01

Full Text Available Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab, L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

Impact of Gene rs7422339 Polymorphism in Argentine Patients With Hyperhomocysteinemia

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Silene M. Silvera-Ruiz BSc

2015-05-01

Full Text Available Carbamoyl phosphate synthetase 1 ( CPS1 is a key gene in the first step of urea cycle and has been correlated with nitric oxide level and vascular smooth muscle activity. A functional single-nucleotide polymorphism C/A at position 4217 in CPS1 (National Center for Biotechnology Information SNP database no. rs7422339, T1405N was reported to be associated with high homocysteine (Hcy plasma values. Although genetic variants of methylenetetrahydrofolate reductase ( MTHFR gene are known to influence Hcy concentration, other genetic determinants of Hcy remain largely unknown. The association between the CPS1 rs7422339 and the risk of hyperhomocysteinemia in Latin American populations is unknown. Here, we study this association in 100 patients having hyperhomocysteinemia without MTHFR c.677C>T polymorphism and 100 controls. CPS1 rs7422339 was studied using polymerase chain reaction and enzymatic restriction. Comparisons of the CPS1 rs7422339 genotype distributions revealed a significant difference between groups ( P = 2.3 × 10 −3 . Patients carrying polymorphic allele showed almost 3 times higher risk (odds ratio [OR] = 2.47 of hyperhomocysteinemia than wild-type allele, suggesting that rs7422339 SNP is associated with high Hcy levels in the Argentine population.

Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL Related Genes and Expression Profiling during Fruit Development in the Chinese Pear

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Yunpeng Cao

2016-10-01

Full Text Available In plants, 4-coumarate:coenzyme A ligases (4CLs, comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR. We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum

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