• BMB Reports
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• v.49 no.12
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• pp.681-686
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• 2016

Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between $1.8{\AA}$ and $2.0{\AA}$. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for $Zn^{2+}$ ion. Citrate and TRIS bound EcFBA structures showed $Zn^{2+}$ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor $Zn^{2+}$ within EcFBA without losing it.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.168-173
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• 1991

The uptake of $Mn^{+2}$, a metal cofactor Mn-SOD, by rice seedings resulted in not only a substantial increase in SOD activity in leaf tissues of the plants, but also a significant enhancement of their cold tolerance : the relative extent of the cold tolerance appeared to accord with relative level of the SOD activity. In contrast, $Fe^{+3},\;Cu^{+2}$ and $Zn^{+2}$, which are the cofactors of Fe-SOD and Cu/Zn-SOD, were found to be ineffective for increasing the SOD activity as well as for improving the chilling-resistant capacity of the plants. The results suggest that Mn-SOD, which is most likely induced by its substrate(superoxide) and activated by the presence of $Mn^{+2}$a at high level, is the enzyme acting as an active component of the defense system against low temperature stress in rice plants. In addition, the application of abscisic acid which has been know to protect to some extent certain plants from chilling injury brought about an increase in SOD activity in rice tissues, providing another affirmative information for the crucial role of SOD under the circumstance of cold stress in plants.

• 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.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.427-434
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• 1998

An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

• KSBB Journal
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• v.15 no.3
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• pp.318-322
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• 2000

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the Enterococcus faecalis RKY1 grown anaerobically on a defined medium containing glycerol and fumarate. A major portion of the purification was performed with employing Triton X-100 and reducing agents by Phenyl-sepharose CL-4B DEAE-sepharose and Dephadex G-150 The final activity was 0.42 unit/mg. The deduced molecular mass of active band was 66 kDa. The optimal pH and temperature for the activity were 7.0 and 38$^{\circ}C$ respectively. The enzyme activity was not affected by 1mM metal ions such as bacl2 $.$2H2O HgCl2 MnCl2$.$4H2O ZnCl2 CuCl2$.$2H2O Mgcl2$.$6H2O FeSo4$.$7H2O and by EDTA. Partially purified enzyme ws yellow in color ; spectroscopic study indicated the presence of flavins as a cofactor.

• BMB Reports
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• v.29 no.1
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• pp.17-21
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• 1996

Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers $Mn^{2+}$ to $Mn^{2+}$ as the cofactor. In $Mn^{2+}$ buffer the mutant is as active as the wild type enzyme in $Mn^{2+}$ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.18-18
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• 2002

Nitrogenase, comprised of the MoFe and Fe proteins, catalyzes the reduction of dinitrogen to ammonia at ambient temperature and pressure. The MoFe protein contains two metal centers, the P-cluster (Fe8S7-8) and the FeMo-cofactor (Fe7S9：homocitrate), the substrate binding site. Despite the availability of the crystal structure of the MoFe protein, suprisingly little is known about the molecular details of catalysis at the active site, and no small-molecule substrate or inhibitor had ever been shown to directly interact with a protein-bound cluster of the functioning enzyme, until our electron-nuclear double resonance(ENDOR) study of CO-inhibited nitrogenase.(omitted)

• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1238-1244
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• 2014

Dephosphocoenzyme A (CoaE) catalyzes the last step in the biosynthesis of the cofactor coenzyme A. In this study, we report the identification and application of CoaE from Stretomyces peucetius ATCC27952. After expression of coaE, the protein was found to have a molecular mass of 28.6 kDa. Purification of the His-tagged fused CoaE protein was done by immobilized metal-affinity chromatography, and then in vitro enzymatic coupling assay was performed. The increasing NADH consumption with time shed light on the phosphorylating activity of CoaE. Furthermore, the overexpression of coaA and coaE independently under the $ermE^*$ promoter in the doxorubicin -producing wild type strain, resulted in 1.4- and 1.5-fold enhancements in doxorubicin production, respectively. In addition, the overexpression of both genes together showed a 2.1-fold increase in doxorubicin production. These results established a positive role for secondary metabolite production from Streptomyces peucetius.

• BMB Reports
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• v.31 no.2
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• pp.130-134
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• 1998

An aldolase gene has been cloned from Methanococcus jannaschii. The coding region of the gene has been expressed in E. coli using a pET system to a level of 30% of total cellular proteins. The protein was purified to more than 95 % homogeneity by heat treatment and ion exchange chromatography. The protein performed an aldol condensation reaction with glyceraldehyde as substrate and dihydroxyacetone phosphate as a carboxyl donor. The protein was determined to be a type II aldolase which requires the $Zn^{2+}$ ion as a metal cofactor. This enzyme has a broad range of optimum pH (7-9) and temperature ($50-80^{\circ}C$). It shows strong stability against heat, chemical denaturants, as well as a high percentage' of organic solvents. The half-life of this enzyme at $85^{\circ}C$ is more than 24 h and it maintains more than 90% of aldolase activity in the presence of 6 M urea, 50% acetonitrile, or 15% isopropyl alcohol.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.