• Molecules and Cells
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• 제39권8호
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• pp.594-602
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• 2016

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

• 한국연초학회지
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• 제20권2호
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• pp.178-182
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• 1998

Previously, we showed that acetone enhanced aryl hydrocarbon hydroxylase (AHH) activity in only 3-methylcholanthrene (MC)- or $\beta$-naphtoflavone (BNF)-inducible microsomes of rat liver. In the present study, the possible mechanism underlying acetone action on AHH was investigated in the liver microsomes from MC-pretreated rats. Other n-alkylketones except acetone did not increase AHH activity, which rather decreased significantly with the length of alkyl side chain. Acetone had no effect on the activity of NADPH-cytochrome P450 reductase or inhibited the formation of 3-OH benzo(a)pyrene (B(a)P) in nonenzymatic model ascorbic acid system. However, in cumene hydroperoxide (CuOOH)-supported B(a)P hydroxylation, acetone enhanced its velocity remarkably by 30% at the optimal concentration (30 $\mu$M CuOOH and 1.0% acetone). From these results, we conclude that acetone may facilitate the formation of an activated oxygen species or the insertion of oxygen into B(a)P molecule in CYP1A rich microsomes.

• 한국환경농학회지
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• 제22권3호
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• pp.203-209
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• 2003

항진균 활성균주를 선발하는 과정에서 DM3 균주를 대천 바닷가에서 수집된 진흙 시료로부터 분리하였으며 API 50CHB kit를 이용하여 동정한 결과 Bacillus licheniformis로 동정되었다. 이 균주는 12종의 식물병원성 진균에 대해 항균활성을 나타내었다. 감마선($^{60}Co$)을 조사하여 항진균 활성 결핍 돌연변이체(mDMB)를 유도한 후, 이차원전기영동으로 단백질을 분석한 결과 DM3와 mDM3에만 존재하는 각각 4종과 3종의 단 단백질을 확인할 수 있었다. 2-DE 결과 B. licheniformis DM3에서 spot 1은 serine hydroxymethyltransferase(45.0kDa), spot 2는 hypothetical protein(40.7 kDa), spot 3는 NifU protein homolog(15.4 kDa), 그리고 spot 4는 resolvase(12.5 kDa)와 상동성을 지닌 단백질로 동정되었고 B. licheniformis mDM3에서만 발현된 spot 5는 lysozyme(18.1 kDa)과 spot 6, 7은 alkyl hydroperoxide reductase(15.6 kDa)으로 동정되었다. B. licheniformis DM3에서 이들 단백질들의 항진균 활성 관련 여부를 규명하기 위해서 더 연구가 진행되어야 할 것으로 사료된다.

• 대한의생명과학회지
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• 제4권1호
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• pp.1-9
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• 1998

효모의 티올특이성 항산화단백과 아미노산 서열상 상동성을 보이는 50종류의 단백은 새로운 항산화 단백군을 형성하며 또한 병원성 미생물에도 널리 분포하고 있으나 이들 단백의 생화학적 및 생리적인 기능은 거의 알려져 있지 않은 실정이다. 본 연구는 병원성 미생물의 티올특이성 항산화단백의 기능에 관한 연구로서 Saccharomyces cerevisiae의 TSA 및 Salmonella typhimurium alkcyl hydroperoxide reductase의 AhpC subunit와 상동성을 나타내는 Corynebacterium diphtheriae의 DirA 유전자를 PCR 방법으로 클로닝하고 대장균에 발현시킨 후 정제하여 항산화 특성을 조사하였다. 정제된 DirA는 티올을 함유하는 금속촉매 산화계인 DTT/Fe$^{3+}$를 선택적으로 억제하였으며 티오레독신 의존성 과산화물 분해활성을 나타내었다. DTT/Fe$^{3+}$ 금속촉매 산화계에 의한 효소의 불활성화를 50% 억제 하는 DirA의 농도는 0.12 mg/ml로 효모 TSA 항산화활성의 약1/4 수준이었으며, 효모의 티 오레 독신계와 반응시켰을때 과산화물 분해활성은 0.02 unit/mg로서 효모 TSA의 티오레독신 의존성 과산화물 분해활성의 1/20수준이었다. 정제된 단백질을 이용하여 항체를 제조하였으며 이항체를 이용하여 Corynebacterium diphtheriae에서 발현됨을 확인하였다. 이러한 결과를 통하여 Corynebacterium diphtheriae의 병원성은 숙주세포의 방어기전인 백혈구에 의하여 생성되는 과산화수소 또는 다른 활성산소종을 제거하는 DirA작용과 연관이 있는 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1380-1389
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• 2015

Prx1, an AhpF-dependent 2-Cys peroxiredoxin (Prx), was previously identified in Vibrio vulnificus, a facultative aerobic pathogen. In the present study, transcription of the V. vulnificus prx1ahpF genes, which are adjacently located on the chromosome, was evaluated by analyzing the promoter and intergenic region of the two genes. Northern blot analyses revealed that transcription of prx1ahpF results in two transcripts, the prx1 and prx1ahpF transcripts. Primer extension analysis and a point mutational analysis of the promoter region showed that the two transcripts are generated from a single promoter. In addition, the 3' end of the prx1 transcript at the prx1ahpF intergenic region was determined by a 3'RACE assay. These results suggested that the prx1ahpF genes are transcribed as an operon, and the prx1 transcript was produced by transcriptional termination in the intergenic region. RNA secondary structure prediction of the prx1ahpF intergenic region singled out a stem-loop structure without poly(U) tract, and a deletion analysis of the intergenic region showed that the atypical stem-loop structure acts as the transcriptional attenuator to result in the prx1 and prx1ahpF transcripts. The combined results demonstrate that the differential expression of prx1 and ahpF is accomplished by the cis-acting transcriptional attenuator located between the two genes and thereby leads to the production of a high level of Prx1 and a low level of AhpF.

• 대한의생명과학회지
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• 제11권4호
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• pp.455-463
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• 2005

Resistance to isoniazid (INH), which is one of the most important drugs in Mycobacterium tuberculosis chemotherapy, has been associated with mutations in genes encoding the mycobacterial catalse-peroxidase (katG), the enoyl acyl carrier protein (ACP) reductase (inhA), alkyl hydroperoxide reductase (ahpC), beta-ketoacyl acyl carrier protein synthase (kasA), and NADH dehydrogenase (ndh). In this study, we examined INH-resistance related genes in 50 INH-resistant and 24 INH-susceptible isolates by PCR-sequence analysis. In brief, mutations at the katG gene were found at codon 315 alone (2/50), at codon 463 alone (19/50), and both at 315 and 463 (29/50). However, while mutations at codon 315 were only detected in INH-resistant isolates, mutations at codon 463 were also detected in INH-susceptible isolates indicating mutations at 463 alone do not seem to confer resistance to INH. Similar to the case of katG 463, some of inhA mutations were also found among INH-susceptible isolates. For example, whereas mutations at 8 upstream of the start codon (UPS) and 15 UPS of the inhA gene were detected only in INH-resistant isolates, mutations at 101, 115, and 125 UPS were detected only in INH-susceptible isolates. Many different kinds of mutations were detected in INH­resistant isolates at ahpC, oxyR gene, and intergenic region of the oxyR-ahpC genes. Howerver, the mutations were not found oxyR and the intergenic regions in INH-susceptible isolates. No mutations were found at either kasA or at ndh gene among INH-resistant isolates. In conclusion, some of mutations such as katG 315, inhA promotor region, and oxyR-ahpC seem to be strongly related to INH-resistance. Currently we are developing a molecular diagnostic method based on these results.