• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1395-1402
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• 2013

Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.

• The Plant Pathology Journal
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• v.29 no.3
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• pp.249-259
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• 2013

Burkholderia glumae causes rice grain rot and sheath rot by producing toxoflavin, the expression of which is regulated by quorum sensing (QS). The QS systems of B. glumae rely on N-octanoyl homoserine lactone, synthesized by TofI and its cognate receptor TofR, to activate the genes for toxoflavin biosynthesis and an IclR-type transcriptional regulator gene, qsmR. To understand genome-wide transcriptional profiling of QS signaling, we employed RNAseq of the wild-type B. glumae BGR1 with QS-defective mutant, BGS2 (BGR1 tofI::${\Omega}$) and QS-dependent transcriptional regulator mutant, BGS9 (BGR1 qsmR::${\Omega}$). A comparison of gene expression profiling among the wild-type BGR1 and the two mutants before and after QS onset as well as gene ontology (GO) enrichment analysis from differential expressed genes (DEGs) revealed that genes involved in motility were highly enriched in TofI-dependent DEGs, whereas genes for transport and DNA polymerase were highly enriched in QsmR-dependent DEGs. Further, a combination of pathways with these DEGs and phenotype analysis of mutants pointed to a couple of metabolic processes, which are dependent on QS in B. glumae, that were directly or indirectly related with bacterial motility. The consistency of observed bacterial phenotypes with GOs or metabolic pathways in QS-regulated genes implied that integration RNAseq with GO enrichment or pathways would be useful to study bacterial physiology and phenotypes.

• BMB Reports
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• v.43 no.6
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• pp.427-431
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• 2010

Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${\beta}1$ and C-terminal ${\alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${\beta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${\alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${\beta}1$ and ${\alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.

• Archives of Pharmacal Research
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• v.29 no.11
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• pp.1049-1054
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• 2006

Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; $100\;{\mu}M\;of\;C_{17}-Sph\;and\;30\;{\mu}g$ protein of F9-12 cells lysate in 20 min. Sphingosine analog $C_{17}-Sph$ was efficiently phosphorylated by Sphk activity ($K_{m}:67.08\;{\mu}M,\;V_{max}\;:1507.5\;pmol/min/mg$). New product $C_{17}-S1P$ was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while $20\;{\mu}M$ of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.

• Horticultural Science & Technology
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• v.29 no.1
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• pp.53-60
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• 2011

Cytoplasmic male sterility (CMS) induced by mutant mitochondria genome, has been used for commercial seed production of $F_1$ hybrid cultivars in diverse crops. In pepper (Capsicum annuum L.), two sterile cytoplasm specific gene organization, atp6-2 and coxII were identified. An open reading frame, orf456 nearby coxII gene has been speculated to induce male sterility (MS) by mutagenic analysis. Moreover, molecular markers for atp6-2 and coxII of mitochondrial genotype (mitotype) were developed. However, the Cytoplasmic MS specific markers, atp6SCAR and coxIISCAR markers appeared in both N and S cytoplasms when polymerase chain reaction (PCR) cycles prolonged more than 40 cycles. Since the reported molecular markers were dominant markers, the presence of the faint sterile-specific band in normal cytoplasm may lead to the mis-classification of pepper breeding lines. To solve this problem, one common forward primer and two different reverse primers specific to normal coxII and sterile orf456 genes were designed after analyzing their gene organizations. By using these three primers, N and S coxII specific bands were co-amplified in male-sterile lines, but only normal coxII specific band was amplified in maintainer lines. Since the reverse primer for sterile coxII was specifically designed 275 bp downstream of orf456, relatively stable PCR amplification patterns were observed regardless of the number of PCR cycles. These primer sets easily identified different mitotypes among the divergent breeding lines, commercial cultivars and diverse germplasms.

• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.

• Bulletin of the Korean Chemical Society
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• v.33 no.8
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• pp.2508-2512
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• 2012

Cold shock proteins (CSPs) are a family of proteins induced at low temperatures. CSPs bind to single-stranded nucleic acids through the ribonucleoprotein 1 and 2 (RNP 1 and 2) binding motifs. CSPs play an essential role in cold adaptation by regulating transcription and translation via molecular chaperones. The solution nuclear magnetic resonance (NMR) or X-ray crystal structures of several CSPs from various microorganisms have been determined, but structural characteristics of psychrophilic CSPs have not been studied. Therefore, we optimized the purification process to obtain highly pure Lm-Csp and determined the three-dimensional structure model of Lm-Csp by comparative homology modeling using MODELLER on the basis of the solution NMR structure of Bs-CspB. Lm-Csp consists of a ${\beta}$-barrel structure, which includes antiparallel ${\beta}$ strands (G4-N10, F15-I18, V26-H29, A46-D50, and P58-Q64). The template protein, Bs-CspB, shares a similar ${\beta}$ sheet structure and an identical chain fold to Lm-Csp. However, the sheets in Lm-Csp were much shorter than those of Bs-CspB. The Lm-Csp side chains, E2 and R20 form a salt bridge, thus, stabilizing the Lm-Csp structure. To evaluate the contribution of this ionic interaction as well as that of the hydrophobic patch on protein stability, we investigated the secondary structures of wild type and mutant protein (W8, F15, and R20) of Lm-Csp using circular dichroism (CD) spectroscopy. The results showed that solvent-exposed aromatic side chains as well as residues participating in ionic interactions are very important for structural stability. Further studies on the three-dimensional structure and dynamics of Lm-Csp using NMR spectroscopy are required.

• Molecules and Cells
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• v.23 no.1
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• pp.23-29
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• 2007

Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-${\gamma}$, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at -399 ~ -388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays.

• Journal of Life Science
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• v.29 no.5
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• pp.537-544
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• 2019

Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).