A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium
Exiguobacterium
sp. Molecular docking analysis ...indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the
k
cat
value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (
K
i,
4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.
GLUL Promotes Cell Proliferation in Breast Cancer Wang, Yanyan; Fan, Shaohua; Lu, Jun ...
Journal of cellular biochemistry,
August 2017, 2017-08-00, 20170801, Letnik:
118, Številka:
8
Journal Article
The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or ...glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs‐based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co‐transfection of Gs‐Enbrel and Aspg‐Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5‐fold higher specific productivity and ~3‐fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.
Nitrogen metabolism in citrus has received increased attention due to its effects on plant growth and productivity. However, little is known about the effects of nitrogen fertilization on nitrogen ...metabolism in young trees of citrus cultivar 'Huangguogan' (Citrus reticulata × Citrus sinensis). Here, genes encoding nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), and asparagine synthetase (AS), represented as HgNR, HgNiR, HgGS, HgGDH, and HgAS, respectively, were cloned from Huangguogan. Deduced protein sequences were analyzed and proteins were confirmed to be localized in their respective cellular organelles. Moreover, pot-cultured 'Huangguogan' seedlings were fertilized with 0 (N1), 1.36 (N2), 1.81 (N3), 2.26 (N4), or 2.72 (N5) kg N/year, for 12 months. Enzyme activity and enzyme-gene expression were studied in roots, leaves, and fruits at different stages. Finally, the effects of N application rate on root activity, leaf N, soluble protein, yield, and fruit quality at the ripening stage were measured. The results showed that: 1) HgNR, HgNiR, HgGDH, and HgAS gene products were found mainly in the cytoplasm and plasma membrane, while HgGS gene product was found mainly in cytoplasm and mitochondria. 2) Gene expression and enzyme activity differed among plant organs. As the root is in permanent direct contact with the soil we suggest that root gene expression and enzyme activity can be used as reference to determine N application rate. 3) Yield, fruit quality, enzyme activity, and enzyme-related gene expression were considerably lower at low than at high-N supply. However, they were all inhibited by excess nitrogen (i.e., 2.72 kg/year). Therefore, we recommend 1.81 kg N/year as the optimal N application rate for young 'Huangguogan' trees.
Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own ...transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.
Hepatocellular adenomas (HCA) with activated β‐catenin present a high risk of malignant transformation. To permit robust routine diagnosis to allow for HCA subtype classification, we searched new ...useful markers. We analyzed the expression of candidate genes by quantitative reverse transcription polymerase chain reaction QRT‐PCR followed by immunohistochemistry to validate their specificity and sensitivity according to hepatocyte nuclear factor 1 alpha (HNF1α) and β‐catenin mutations as well as inflammatory phenotype. Quantitative RT‐PCR showed that FABP1 (liver fatty acid binding protein) and UGT2B7 were downregulated in HNF1α‐inactivated HCA (P ≤ 0.0002); GLUL (glutamine synthetase) and GPR49 overexpression were associated with β‐catenin–activating mutations (P ≤ 0.0005), and SAA2 (serum amyloid A2) and CRP (C‐reactive protein) were upregulated in inflammatory HCA (P = 0.0001). Immunohistochemistry validation confirmed that the absence of liver‐fatty acid binding protein (L‐FABP) expression rightly indicated HNF1α mutation (100% sensitivity and specificity), the combination of glutamine synthetase overexpression and nuclear β‐catenin staining were excellent predictors of β‐catenin–activating mutation (85% sensitivity, 100% specificity), and SAA hepatocytic staining was ideal to classify inflammatory HCA (91% sensitivity and specificity). Finally, a series of 93 HCA was unambiguously classified using our 4 validated immunohistochemical markers. Importantly, new associations were revealed for inflammatory HCA defined by SAA staining with frequent hemorrhages (P = 0.003), telangiectatic phenotype (P < 0.001), high body mass index, and alcohol intake (P ≤ 0.04). Previously described associations were confirmed and in particular the significant association between β‐catenin–activated HCA and hepatocellular carcinomas (HCC) at diagnosis or during follow‐up (P < 10−5). Conclusion: We refined HCA classification and its phenotypic correlations, providing a routine test to classify hepatocellular adenomas using simple and robust immunohistochemistry. (HEPATOLOGY 2007.)
•Overexpression studies using GS1 to increase plant nitrogen use efficiency (NUE) have not yielded consistent results.•Complex regulation of GS1 at gene and protein levels complicates efforts to ...improve plant NUE use efficiency by GS1 overexpression.•We suggest future strategies for the use of GS1 to increase NUE.
Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude that GS1 activity may be downregulated via a chain of processes elicited by metabolic imbalances and environmental constraints. We suggest that a pivotal role of GS1 may be related to the maintenance of essential nitrogen (N) flows and internal N sensing during critical stages of plant development. A number of more refined overexpression strategies exploiting gene stacking combined with tissue and cell specific targeting to overcome metabolic bottlenecks are considered along with their potential in relation to new N management strategies.
Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for ...glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.
Tuberculosis remains a major health problem accentuated by the rise of resistance to all available drugs. Therefore, this study was launched to discover a novel antituberculosis agent from wild ...Egyptian Sahara plants. Twelve such plants were screened, in vitro, for their activity against various Mycobacterium species. The most active plant, Euphorbia paralias, was further fractionated with different organic solvents, and the activity of the obtained fractions was determined by the agar diffusion and broth microdilution methods. The methanol fraction was the most active against Mycobacterium spp., and was non-toxic in doses up to 10 g/kg of animal weight. Its main component was separated by column chromatography, and then identified by ultraviolet spectroscopy and nuclear magnetic resonance analysis as quercetin-3-O-β-D-glucoside. Docking analysis suggested that quercetin-3-O-β-D-glucoside inhibits the glutamine synthetase enzyme, a promising target for the development of antituberculosis drugs. This prediction was confirmed by an in vitro glutamine synthetase biosynthetic assay. To the best of our knowledge, and based on bioinformatics mining of the BioPhytMol database, this is the first report on the antimycobacterial activity of Euphorbia paralias plant. It is also the first report on the inhibition of mycobacterial glutamine synthetase by the flavonoid quercetin.
Summary
Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which ...external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs‐silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one‐hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN‐1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO‐mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.
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