• BMB Reports
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• v.40 no.5
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• pp.649-655
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• 2007

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires $Mg^{2+}$ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.

• BMB Reports
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• v.40 no.2
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• pp.239-246
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• 2007

Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a $C_2$-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant $^{19}F$ NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.

• BMB Reports
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• v.33 no.4
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• pp.317-320
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• 2000

The succinic semialdehyde dehydrogenase from bovine brain was inactivated by treatment with phenylglyoxal, a reagent that specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics with an apparent secondorder rate constant of 30 $M^{-1}min^{-1}$ for inactivation. Partial protection against inactivation was provided by the coenzyme $NAD^+$, but not by the substrate succinic semialdehyde. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the binding of 2 mol phenylglyoxal per mol of enzyme. These results suggest that essential arginine residues, located at or near the coenzyme-binding site, are connected with the catalytic activity of brain succinic semialdehyde dehydrogenase.

• BMB Reports
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• v.29 no.1
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• pp.32-37
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• 1996

The cysteine 43, potentially important in the activity of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium, has been changed to serine. This mutant enzyme (C43S) has been studied in order to gain insight into the structural basis for the binding of inhibitor, substrate and product. UV-visible spectra of C43S exhibit the same spectral change in the presence of OAS as that observed with wild type enzyme, indicating C43S will form an ${\alpha}$-aminoacrylate Schiff base intermediate. At pH 6.5, however, the deacetylase activity of C43S is much higher than wild type enzyme indicating that cysteine 43 plays a role in stabilizing the ${\alpha}$-aminoacrylate intermediate. The fluoroscence spectrum of C43S exhibits a ratio of emission at 340 to 502 nm of 16.9, reflecting the lower fluorescence of PLP and indicating that the orientation of cofactor and tryptophan are different from that of the wild type enzyme. The emission spectrum of C43S in the presence of OAS gives two maxima at 340 and 535 nm. The 535 nm emission is attributed to the fluoroscence of the ${\alpha}$-aminoacrylate intermediate. The visible circular dichroic spectrum was similar to wild type enzyme, but the negative effect observed at 530~550 nm and the molar ellipicity values for the mutant are decreased by about 50% compared to wild type enzyme. The circular dichroic and fluoroscence studies suggest binding of the cofactor is less asymmetric in C43S than in the wild type enzyme.

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.65-75
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• 2015

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to $Zn^{2+}$ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.2
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• pp.56-60
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• 2016

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of $^1H-^{15}N$ HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.89-97
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• 2015

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40℃, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k_cat/K_m) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45℃, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176^th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca²⁺, whichincreased the thermostability of M179.

• Proceedings of the Korean Vacuum Society Conference
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• 2016.02a
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• pp.146-146
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• 2016

Zinc Oxide (ZnO) was known as a promising material for surface acoustic wave devices, gas sensors, optical devices and solar cells due to piezoelectric material, large band gap of 3.37 eV and large exciton binding energy of 60 meV at room temperature. In particular, the alignment of ZnO nanostructures into ordered nanoarrays can bring about improved sensitivity of devices due to widen the surface area to catch a lot of gas particle. Oxygen plasma treatment is used to specify the nucleation site of round patterned ZnO nanorods growth. Therefore ZnO nanorods were grown on a quartz substrate with patterned polystyrene monolayer by hydrothermal method after oxygen plasma treatment. And then, we carried out nanostructures by adjusting the diameter of the arranged ZnO nanorods according to polystyrene spheres of various sizes. The obtained ZnO nanostructures was characterized by X-ray diffraction (XRD), Field emission scanning electron microscopy (FE-SEM).

• BMB Reports
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• v.28 no.1
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• pp.40-45
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• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.636-642
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• 1994

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.