• Journal of environmental and Sanitary engineering
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• v.23 no.2
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• pp.1-18
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• 2008

The tetrachloroethylene (PCE) dehalogenase of Clostridium bifermentans DPH-1 (a halorespiring organism) was purified, cloned, and sequenced. This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of dichloroethylene isomers along with PCE and trichloroethylene (TCE). Broad range of substrate specificity for chlorinated aliphatic compounds (PCE, TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropene, and 1,1,2-trichloroethane) for this enzyme was also observed. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. A partial sequence (81 bp) of the encoding gene for PCE dehalogenase was amplified and sequenced with degenerateprimers designed from the N-terminal sequence (27 amino acid residues). Southern analysis of C. bifermentans genomic DNA using the polymerase chain reaction product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. So, C. bifermentans could play some important role in the initial breakdown of PCE and other chlorinated aliphatic compounds in sites contaminated with mixtures of halogenated substances.

• BMB Reports
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• v.42 no.9
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• pp.586-592
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• 2009

The bactenecin is an antibacterial peptide with an intramolecular disulfide bond. We recently found that homodimeric bactenecin exhibits more potent antibacterial activity than the monomeric form and retains its activity at physiological conditions. Here we assess the difference in the modes of antibiotic action of homodimeric and monomeric bactenecins. Both monomeric and dimeric bactenecins almost completely killed both Staphylococcus aureus and E. coli within 10-30 min at concentrations of $8-16\;{\mu}M$. However, exposure to liposomes elicited an increase in the fluorescence quantum yield from a tryptophan-containing monomeric analog, while the homodimeric analog showed a significant reduction in fluorescence intensity. Moreover, unlike the monomer, the homodimer displayed apparent membrane-lytic activity enabling release of various sized dyes from liposomes, and rapidly and fully depolarized the S. aureus membrane. Together, our results suggest that homodimeric bactenecin forms pores in the bacterial membrane, while monomeric one penetrates through the membrane to target intracellular molecules/organelles.

• BMB Reports
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• v.36 no.5
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• pp.442-449
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• 2003

NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction. We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones. The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized. The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide. The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868. The $M_r$ that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer. The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones. These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.

• 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.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.928-937
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• 2016

S-Nitrosoglutathione reductase (GSNOR) metabolizes S-nitrosoglutathione (GSNO) and has been shown to play important roles in regulating cellular signaling and formulating host defense by modulating intracellular nitric oxide levels. The enzyme has been found in bacterial, yeast, mushroom, plant, and mammalian cells. However, to date, there is still no evidence of its occurrence in filamentous fungi. In this study, we cloned and investigated a GSNOR-like enzyme from the filamentous fungus Aspergillus nidulans. The enzyme occurred in native form as a homodimer and exhibited low thermal stability. GSNO was an ideal substrate for the enzyme. The apparent K_m and k_cat values were 0.55 mM and 34,100 min^-1, respectively. Substrate binding sites and catalytic center amino acid residues based on those from known GSNORs were conserved in this enzyme, and the corresponding roles were verified using site-directed mutagenesis. Therefore, we demonstrated the presence of GSNOR in a filamentous fungus for the first time.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1096-1104
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• 2014

$\small{L}$-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of $\small{L}$-asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was $37^{\circ}C$ and over 90% of activity was retained between $37^{\circ}C$ and $50^{\circ}C$, and its thermal stability range was narrower than that of commercial E. coli $\small{L}$-asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The $K_m$ value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.

• Reproductive and Developmental Biology
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• v.39 no.4
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• pp.105-115
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• 2015

Toll-like receptor 7 (TLR7) is critical for the triggering of innate immune response by recognizing the conserved molecular patterns of single-stranded RNA (ssRNA) viruses and mediated antigenic adaptive immunity. To understand how TLR7 distinguish pathogen-derived molecular patterns from the host self, it is essential to be able to identify TLR7 receptor interaction interfaces, such as active sites or R848-agonist binding sites. The functional interfaces of TLR7 can serve as targets for structure-based drug design in studying the TLR7 receptor's structure-function relationship. In contrast to mammalian TLR7, chicken TLR7 (chTLR7) is unknown for its important biological function. Therefore, it has been targeted to mediate contrasting evolutionary patterns of positive selection into non-synonymous SNPs across eleven species using TLR7 conservation patterns (evolutionary conserved and class-specific trace residues), where protein sequence differences to the TLR7 receptors of interest record mutation that have passed positive section across the species. In this study, we characterized the Lys609 residue on chTLR7-ECD homodimer interfaces to reflect the current tendency of evolving positive selection to be transfer into a stabilization direction of the R848-agonist/chTLR7-ECDs complex under the phylogenetically variable position across species and we suggest a potential indicator for contrasting evolutionary patterns of both the species TLR-ECDs.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1070-1083
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• 2015

A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His⁴⁰⁰-Glu⁴⁰¹-X-XHis⁴⁰⁴). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepF_Ba from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the K_m, V_max, and k_cat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10^-6 M, 2.65 ± 0.03 × 10^-3 µM/min, and 5.99 ± 0.07 s^-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.

• IMMUNE NETWORK
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• v.3 no.4
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• pp.276-280
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• 2003

Background: Peroxidases (Prx) of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol respectively. Hydrogen peroxide is implicated as an intracellular messenger in various cellular responses such as proliferation and differentiation. And Prx I activity is regulated by Cdc-2 mediated phosphorylation. This work was undertaken to investigate the proliferation role of peroxiredoxin III as a member of Prx family in Prx III overexpressed HeLa cell line. Methods: To provide further evidence of proliferation, we selected Prx III stably expressed HeLa Tet-off cell lines. Cell proliferation was examined by using proliferation reagent WST-1 in the presence or absence of doxycycline. Prx III, 2-cys Prx enzymes exist as homodimer. The activation of Prx III heterodimer with induced and endogenous Prx III was examined by immunoprecipitation. Results: Immunoprecipitation analysis of the induced and endogenous Prx III with anti-myc showed that the induced wild type (WT) and dominant negative (DN) Prx III from HeLa Prx III Tet-off stable cell heterodimerized with endogenous Prx III each other. And the expression level of induced Prx III was examined after addition of doxycycline. By 72 hr, the expression level of induced Prx III was diminished gradually and the half-life of the induced wild type Prx III was approximately 17 hr. The proliferation experiment demonstrated that the relative proliferation value of induced and endogenous WT Prx III stable cell has no changes but the DN Prx III induced HeLa Tet-off stable cells were lower than endogenous Prx III. Conclusion: In conclusion, the HeLa dominant negative Prx III Tet-off stable cells were decreased the proliferation.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.108-108
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• 2002

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.