• BMB Reports
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• v.34 no.2
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• pp.172-175
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• 2001

Catechol 1,2-dioxygenase $I_1$ ($CDI_1$) is the first enzyme of the $\beta$-ketoadipate pathway in Acinetobacter lowffii K24. $CDI_1$ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, $AgNO_3$. Two mutants, $CDI_1$ C155V and $CDI_1$ C202V, were obtained by site-directed mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys$\rightarrow$Val) were characterized by peptide mapping and amino acid sequencing. Interestingly, $CDI_1$ C155V was inhibited by $AgNO_3$, whereas $CDI_1$ C202V was not inhibited. This suggests that $Cys^{202}$ is the sole inhibition site by $AgNO_3$ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated $CDI_1s$ suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of $CDI_1$.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.381-388
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• 2001

The present study was designed to obtain porcine growth hormone binding protein (pGHBP) improved biological activation as derived mutation in binding site with growth horlnone (GH). A 756 bp of fragment encoding the extracellular domain of pGHBP gene was cloned from the total RNA of porcine fat tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and created mutation in positions 26 and 122 using site-directed mutagenesis method. Position 26 is one and it is near to get on five potential N-linked glycosylation sites located in the extracellular domain of porcine growth hormone receptor known to have a direct influence on combination with GH. Position 122 is known as one of conformational epitope in bovine. It was over-expressed in E. coli using pET-32(c) expression vector and precisely purified by S-protein agarose and enterokinase. In our results, we was obtained pmGHBP of 30 kDa. It suggests to study the effects of the pmGHBP on cell proliferation in vitro and growth rate in vivo after administration.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.823-827
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• 2003

Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolactones. It has been reported that the generation of two macrolactone structures results from alternative expression of pikromycin (Pik) polyketide synthase (PKS). It was previously reported that the hybrid pikromycin-tylosin PKS can also produce two different macrolactones but its mechanistic basis remains unclear. In order to address this question, a series of site-directed mutagenesis of tentative alternative ribosome binding site and translation start codons in tylGV were performed. The results suggest that macrolactone ring size is not determined by the alternative expression of TylGV but through other mechanism(s) involving direct interaction between the PikAIII and TE domain or skipping of the final chain elongation step. This provides new insight into the mechanism of macrolactone ring size determination in hybrid PKS as well as an opportunity to develop novel termination activities for combinatorial biosynthesis.

• BMB Reports
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• v.29 no.6
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• pp.513-518
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• 1996

A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiolcontaining oxidation system but not against an oxidation system without thiol. This 25-kDa protein was thus named thiol-dependent protector protein (TPP). The role of TPP in the cellular defense against oxidative stress was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPP (strain YP) and a mutant in which the catalytically essential amino acid in the active site of TPP (Cys-47) has been replaced with alanine by site-directed mutagenesis (strain YPC47A). There was a distinct difference between these two strains in regard to viability, modulation of activities of superoxide dismutase and catalase, and the oxidative damage of DNA upon exposure to menadione. These results suggest that TPP may play a direct role in the cellular defense against oxidative stress by functioning as an antioxidant protein.

• Journal of the Korean Electrochemical Society
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• v.7 no.1
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• pp.26-31
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• 2004

The direct electrochemical studies of four laccases (plant and fungal laccases) have been investigated on a gold electrode functionalized with a new tether of 2.2'-dithiosalicylic aldehyde. Results from these studies indicate that the redox potential of the active site of plant laccase from Rhus vernificera is shifted to a more negative value(255 mV versus SCE) than that of fungal laccase from Pyricularia oryzae (480 mV versus SCE). Mechanistic studies indicate that the reduction of type-1 Cu precedes the reduction of type-2 and type-3 Cu ions when the electrode is poised initially at different potentials. Also a new tether, 2.2'-dithiosalicylic aldehyde, has been used to study the redox properties of two laccases (LCCI and Lccla) covalently attached to a gold electrode. An irreversible peak at 0.47V vs. SCE is observed in the cyclic voltammorams of LCCI. In contrast, the cyclic voltammograms of LCCIa contain a quasi-reversible peak at 0.18V vs. SCE and an irreversible peak at 0.50V vs. SCE. We find that the replacement of the eleven amino acids a the C-terminus with a single cysteine residue $(i.e., \;LCCI{\rightarrow}LCCIa)$ influences the rate of heterogeneous electron transfer between an electrode and the copper containing active sites $(K_{het}\;for\;LCCI=1.0\times10^{-2}\;s^{-1}\;and\;K_{het}\;for\;LCCI_a= 1.0\;times10^{-1}\;s^{-1}\'at\;0.18V\;versus\;SCE\;and\;4.0\times10^{-2}\;s^{-1}\;at\;0.50V\; versus\;SCE)$. These results show for the first time that the change of the primary structure of a protein via site-directed mutagenesis influences both the redox potentials of the copper ions in the active site and the rate of heterogeneous electron transfer.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.79-86
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• 2012

ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.353-360
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• 2003

For the simple rapid bioassay of organophosphorus and carbamate pesticide residues, a mass-production system of acetycholinesterase (AChE, EC 3.1.1.7, MAChE) using baculovirus and insect cell culture was constructed. The cDNA for AChE was synthesized from Drosophila melanogaster in Halla Mountain, the lipid anchor tail was removed by PCR and was used for the site-directed mutagenesis of three amino acid residues (E107Y, F368L, L408F). The mutated cDNA was inserted into the baculovirus vector and expressed in insect cells. Maximum cell growth and enzyme activity were reached when the cells $(2{\times}10^6\;cell/ml)$ were infected four times at four-day-intervals. His-tag containing MAChE was purified using Ni-NTA column and used for characterization. The activity was maintained under various pHs (3-10) and temperatures $(20-50^{\circ}C)$ under experimental conditions. As an extraction solution for pesticides, methanol is more effective than ethanol. Against major organophosphate and carbamate pesticides, the MAChE showed better sensitivity than AChE and AChE from housefly (Taiwan).

• Molecules and Cells
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• v.44 no.10
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• pp.723-735
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• 2021

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.1
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• pp.91-99
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• 2018

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, $^{922}FMDRLK^{927}$, in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922-927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the ${HCO_3}^-$ transport. These results suggested that like IRBIT, PP1 was another novel regulator of ${HCO_3}^-$ secretion in several types of epithelia.

• BMB Reports
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• v.46 no.4
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• pp.219-224
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• 2013

The cell-fate specification of the anchor cell (AC) and a ventral uterine precursor cell (VU) in Caenorhabditis elegans is initiated by a stochastic interaction between LIN-12/Notch receptor and LAG-2/Delta ligand in two neighboring Z1.ppp and Z4.aaa cells. Both cells express lin-12 and lag-2 before specification, and a small difference in LIN-12 activity leads to the exclusive expressions of lin-12 in VU and lag-2 in the AC, through a feedback mechanism of unknown nature. Here we show that the expression pattern of lag-1/CSL, a transcriptional repressor itself that turns into an activator upon binding of the intracellular domain of Notch, overlaps with that of lin-12. Site-directed mutagenesis of LAG-1 binding sites in lag-1 maintains its expression in the AC, and eliminates it in the VU. Thus, AC/VU cell-fate specification appears to involve direct regulation of lag-1 expression by the LAG-1 protein, activating its transcription in VU cells, but repressing it in the AC.