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Discovery and Characterization of a Thermostable NADH Oxidase from Pyrococcus horikoshii OT3
Jong-Uk Koh,Hyun-Jung Chung,Woo-Young Chang,Masaru Tanokura,공광훈 대한화학회 2009 Bulletin of the Korean Chemical Society Vol.30 No.12
A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced β-NADH with subsequent formation of H2O2 in the presence of FAD as a cofactor, but not α-NADH, α-NADPH, or β-NADPH. PhNOX showed high affinity for β-NADH with a Km value of 3.70 μΜ and exhibited optimum activity at pH 8.0 and 95 oC as it is highly stable against high temperature.
Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1
Koh, Jong-Uk,Cho, Hyun-Young,Kong, Kwang-Hoon Korean Chemical Society 2007 Bulletin of the Korean Chemical Society Vol.28 No.5
In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. Km GSH, Km DCNB and Km EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.
Discovery and Characterization of a Thermostable NADH Oxidase from Pyrococcus horikoshii OT3
Koh, Jong-Uk,Chung, Hyun-Jung,Chang, Woo-Young,Tanokura, Masaru,Kong, Kwang-Hoon Korean Chemical Society 2009 Bulletin of the Korean Chemical Society Vol.30 No.12
A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced ${\beta}$-NADH with subsequent formation of $H_2O_2$ in the presence of FAD as a cofactor, but not ${\alpha}$-NADH, ${\alpha}$-NADPH, or ${\beta}$-NADPH. PhNOX showed high affinity for ${\beta}$-NADH with a Km value of 3.70 ${\mu}$M and exhibited optimum activity at pH 8.0 and 95$^{\circ}C$ as it is highly stable against high temperature.
Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1
Jong-Uk Koh,Hyun-Young Cho,공광훈 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.5
In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. KmGSH, KmDCNB and KmEPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.
조백기,박종갑,김형옥,김성욱,백승철,김진우,김시용,안규중,전재복,오지원,김낙인,이규석,오칠환,김수남,김상태,손숙자,신용우,김동석,이원우,권경술,서대헌,황규왕,이종석,고재경,강원형,정기양,최응호,김기홍,박석돈,강승주,함정희,명기범,김방순,구상완,김병수,원영호,김한욱,송은섭,정병수,노병인,홍창권,박장규,한지윤,김광중,구대원,김종민,김재홍,유희준,양경미 대한의진균학회 1998 대한의진균학회지 Vol.3 No.2
Background: Onychomycosis, especially toenail onychomycosis has become one of the common fungal infection and has historically been regarded as a cosmetic rather than medical problem by many patients, even by physicians. Recently, however, there are several reports that is a refractory disease which may cause a deleterious effect on patiets quality of life (QOL). Objective: The purpose of this study was to investigate the impact of toenail onychomycosis on QOL in Korea and to assess the chandes of QOL after treatment. Methods: Total 1004 patients with toenail onychomycosis which was confirmed by clinical findings and KOH preparation were were enrolled at 47 dermatologic centers in Korea, and interviewed with standardized QOL questionnaire before and after sytemic antifungal treatment. Responses to the questionnaire were scored by 5-point scale (1∼4) and averaged, and were analyzed for 5 dimensions of emotional impact, social impact, symptorn and functional impact, patients views concerning treatment, and relationship with doctor. Results: 1. Before and after treatment, the most serious impact was emotional dimension showing 1.90 and 1.30 in average score (AS), and social (AS: 1.14 and 0.83) and symptom and functional impact (AS: 1.05 and 0.92) was also affected. 2. In female rather than male, statistically more significant impact on patients QOL was observed in all dimensions. 3. After treatment, 3 of 5 dimensions were improved significantly - emotional dimension (AS : from 1.90 to 1.30), social dimension (AS: from 1.14 to 0.83), patients view concerning treatment (AS: from 1.34 to 1.02). 4. The drgree of patients satisfaction at the therapeutic effect was very high- 62.4% (immediately after treatment) and 65.8% (9 months affer initiation of treatment) of patients answered excellent or good. Conclusion: This study confirms that toenail onychomycosis has significant impact on the overall QOL of patients. Also the effect of antifungal therapy on patients QOL were watisfactory. Therefore, both doctor and patient should pay more attention to the treatment of onychomycosis. [Kor J Med Mycol 3(1): 115∼124]