• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.186-195
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• 2015

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive ${\alpha}$ subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

• BMB Reports
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• v.32 no.4
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• pp.393-398
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• 1999

Acetolactate synthase (ALS) catalyzes the first reaction common to the biosynthesis of L-valine, L-leucine, and L-isoleucine. ALS is the target site of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Two forms of ALS (ALS I and ALS II) which have different affinity for Heparin have been separated from etiolated pea seedlings. The substrate saturation curves of both ALS I and ALS II were hyperbolic in contrast to previous reports. The two forms of ALS showed significant differences in their physical and kinetic properties. The values of $K_m$ for ALS I and ALS II were 9.0 mM and 4.8 mM, respectively. The pI values for ALS I and ALS II were determined to be 5.3 and 5.75 by isoelectric focusing, respectively. The native molecular weights for ALS I and ALS II obtained by nondenaturing gel electrophoresis and activity staining were 124 and 244 kDa, respectively. They also exhibited different sensitivity to feedback inhibition by end-product amino acids and inhibition by Cadre, an imidazolinone herbicide.

• BMB Reports
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• v.50 no.1
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• pp.1-2
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• 2017

Acquiring a selective growth advantage by breaking the proliferation barrier established by gatekeeper genes is a centrally important event in tumor formation. Removal of the mammalian Hippo kinase Mst1 and Mst2 in hepatocytes leads to rapid hepatocellular carcinoma (HCC) formation, indicating that the Hippo signaling pathway is a critical gatekeeper that restrains abnormal growth in hepatocytes. By rigorous genetic approaches, we identified an interacting network of the Hippo, Wnt/${\beta}$-catenin and Notch signaling pathways that control organ size and HCC development. We found that in hepatocytes, the loss of Mst1/2 leads to the activation of Notch signaling, which forms a positive feedback loop with Yap/Taz (transcription factors controlled by Mst1/2). This positive feedback loop results in severe liver enlargement and rapid HCC formation. Blocking the Yap/Taz-Notch positive feedback loop by Notch inhibition in vivo significantly reduced the Yap/Taz activities, hepatocyte proliferation and tumor formation. Furthermore, we uncovered a surprising inhibitory role of Wnt/${\beta}$-catenin signaling to Yap/Taz activities, which are important in tumor initiation. Genetic removal of ${\beta}$-catenin in the liver of the Mst1/2 mutants significantly accelerates tumoriogenesis. Therefore, Wnt/${\beta}$-catenin signaling, known for its oncogenic property, exerts an unexpected function in restricting Yap/Taz and Notch activities in HCC initiation. The molecular interplay between the three signaling pathways identified in our study provides new insights in developing novel therapeutic strategies to treat liver tumors.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.273-277
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• 1997

The sesquiterpene cyclase (SC) was induced and its products were accumulated in the culture media of Hyoscyamus muticus hairy roots by addition of Rhizoctonia solani extracts. The cumulative production of solavetivone was nearly doubled by integrated extraction of the products from the media during the 24 h accumulation period. Western blots with monoclonal antibodies against SC show that the enzyme levels are the same for both extracted and non-extracted cultures. SC activities measured in vitro with radioactive substrate are not significantly different. These results suggest that productivity is controlled by substrate availability within the terpenoid pathway, and feedback regulation precedes the branch-point enzyme sesquiterpene cyclase.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.506-508
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• 1986

Metabolic activity of inorganic nitrogenous compounds affects not only microbial growth but also metabolite production in fermentation technology. We have worked on the enzymes participating in ammonia assimulation of some fermentation bacteria. This paper summarizes the results on glutamine synthetase and its application in practical field. Glutamine synthetase (L-glutamate:ammonia ligase, EC. 6.3.1.2) catalyzes the formation of glutamine from glutamate and ammonia at the expense of cleavage of ATP and inorganic phosphate. The enzyme plays a dual role in nitrogen metabolism in bacteria; it is a key enzyme not only in the biosynthesis of various compounds through glutamine but also in the regulation of synthesis of some enzymes involved in the metabolism of nitrogenous compounds. The detailed works with the Eschericia coli and other enterobacterial enzymes revealed that glutamine synthetase is controlled by the following complex of mechanisms: (a) feedback inhibition by end products, (b) repression and derepression of enzyme synthesis, (c) modulation of enzyme activity in response to divalent cation and (d) covalent modification of enzyme protein by adenylylation and its cascade control. Comparative studies have also been made on the enzymes from other organisms.

• BMB Reports
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• v.34 no.4
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• pp.299-304
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• 2001

The 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase is the first enzyme of aromatic amino acid-, folic acid-, and phenazine-1-carboxylic acid biosynthetic pathways. DAHP synthase of Bacillus sp. B-6 that produces phenazine-1-carboxylic acid was feedback inhibited by two intermediary metabolites of aromatic amino acid biosynthetic pathways, prephenate and chorismate, but not by other metabolites, such as anthranilic acid, shikimic acid, p-aminobenzoic acid, and 3-hydroxyanthranilic acid. DAHP synthase of Bacillus sp. B-6 was not inhibited by end products, such as aromatic amino acids, folic acid, and phenazine-1-carboxylic acid. The inhibition of DAHP synthase by prephenate and chorismate was non-competitive with respect to erythrose 4-phosphate and phosphoenolpyruvate. Prephenate and chorismate inhibited 50% of the DAHP synthase activity at concentrations of $2{\times}10^{-5}\;M$ and $1.2{\times}10^{-4}\;M$, respectively The synthesis of DAHP synthase of Bacillus sp. B-6 was not repressed by exogenous aromatic amino acids, folic acid, and phenazine 1-carboxylic acid, single or in combinations.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.254-261
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• 2012

The purpose of this research was to optimize electric current production of sediment microbial fuel cells by injecting glucose and to investigate its impact on microbial communities involved. It was shown that injection of proper concentration of glucose could increase electric current generated from sediment microbial fuel cells. When 1,000 mg/L of glucose, as opposed to higher concentrations, was injected, electric current increased up to 3 times. This increase is mainly attributed to the mutual relationship between fermenting bacteria and exoelectrogenic bacteria. Here the organic acids generated by fermenting bacteria could be utilized by exoelectrogenic bacteria, removing feedback inhibition caused by the organic acids. When glucose was injected, the population of Clostridium increased as to ferment injected glucose. Glucose fermentation can have either a positive or negative effect on electric current generation. When exoelectrogenic bacteria may readily utilize the end-product, electric current could increase. However, when the end-product was not readily removed, then detrimental chemical reactions (pH decrease, methane generation, organic acids accumulation) occurred: exoelctrogenic bacteria population declined and non-microbial fuel cell related microorganisms prospered. By injecting a proper concentration of glucose, a mutual relationship between fermenting bacteria, such as Clostridium, and exoelectrogenic bacteria, such as Geobacter, should be fulfilled in order to increase electricity production in mixed cultures of microorganisms collected from the aquatic sediments.

• Journal of the Korean Chemical Society
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• v.39 no.6
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• pp.459-465
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• 1995

Acetolactate Synthase (ALS) was partially purified from the yeast and its basic biochemical studies were carried out. Yeast was grown in the minimum media containing 0.5% glucose, 51 mM $K_2HPO_4$, 22 mM $KH_2PO_4$, 8 mM $(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$ for 18 hours at 37 $^{\circ}C$. The cell was ruptured in the buffer (20 mM phosphate buffer pH 7.0, 0.1 mM TPP, 0.5 mM DTT, 1 ${\mu}M$ FAD, and 1 mM MgCl_2$) following an overnight suspension. The supernatant fraction was collected from $10,000{\times}g$ and the enzyme was further purified by ammonium sulfate fractionation, DEAE-Sephacel chromatography and leucine-agarose chromatography. The enzyme activity was measured under the various conditions by the function of protein concentration, time, temperature, pH, and substrate. The optimum temperature was found to be 50$^{\circ}C$, optimum pH 8.0∼8.5. The kinetic parameters, $K_m\;and\;V_{max}$ were 8.4 mM and 17.9 nmol/mg/min respectively. Stability of the enzyme was studied with ethylene glycol and glycerol added to the enzyme solution. Both ethylene glycol and glycerol improved the enzyme stability up to 50%. The study of feedback inhibition showed that valine was a strong inhibitor while leucine was a weak inhibitor.

• Korean Journal of Head & Neck Oncology
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• v.30 no.2
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• pp.53-61
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• 2014

Background and Objectives : Anaplastic thyroid carcinoma(ATC) is a rare but highly aggressive thyroid malignancy that is associated with an extremely poor survival despite the best multidisciplinary care. BRAF(V600E) mutation is detected in about a quarter of ATC, but unlike its high treatment response to selective BRAF inhibitor (PLX4032) in metastatic melanoma, the treatment response of ATC is reported to be low. The purpose of this study is to investigate the innate resistance mechanism responsible for this low treatment response to BRAF inhibitor and its effect on epithelial-mesenchymal transition(EMT). Materials and Methods : Two ATP cell lines, 8505C and FRO were selected and treated with PLX4032 and its drug sensitivity and effects on cell migration and EMT were examined and compared. Further investigation on the changes in signals responsible for the different treatment response to PLX4032 was carried out and the same experiment was performed on both orthotopic and ectopic xenograft mouse models. Results : FRO cell line was more sensitive to PLX4032 treatment compared to 8505C cell line. The resistance to BRAF inhibition in 8505C was due to increased expression of EGFR. Effective inhibition of both EGFR and p-AKT was achieved after dual treatment with BRAF inhibitor(PLX4032) and EGFR inhibitor(Erlotinib). Similar results were confirmed on in vivo study. Conclusion : EGFR-mediated reactivation of the PI3K/AKT pathway and MAPK pathway contributes to the relative insensitivity of BRAF(V600E) mutant ATC cells to PLX4032. Dual inhibition of BRAF and EGFR leads to sustained treatment response including cell invasiveness.