• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1750-1759
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• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.109-109
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• 2001

The present study was done to investigate the relationship between Kupffer cells and alteration of cytochrome P-450 (CYP)-dependent drug metabolizing enzyme activities during polymicrobial sepsis. Male rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. The gadolinium chloride (GdC1$_3$, 10 mg/kg), blocker of Kupffer cells, was pretreated intravenously at 48 h and 24 h prior to the induction of CLP. All assay parameters were determined at 24 h after CLP or sham operation. In CLP-treated rats, the mortality rate of animals increased to 50% and serum alanine (ALT) and aspartate aminotransferase (AST) levels also significantly elevated. However, this increase was not suppressed by GdC1$_3$ pretreatment. Microsomal lipid peroxidation markedly increased after CLP operation. This increase was significantly attenuated by pretreatment. Total cytochrome P-450 content and NADPH-cytochrome P-450 reductase activity were not changed after CLP operation, but GdC1$_3$pretreatment reduced total cytochrome P-450 content, The hepatic microsomal CYP 1A1, 1A2, 2Bl and 2El activities in CLP-induced rats were also not significantly different from sham-operated rats. However, GdC1$_3$pretreatment showed a moderate increase in CYP1A1 and 1A2 activities. Our findings suggest that Kupffer cells may be partly responsible for producing hepatocellular dysfunction during sepsis.

• 약학회지
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• 제43권6호
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• pp.834-842
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• 1999

The metabolism and pharmacokinetics of M-l, which is metabolite of pentoxifylline, have been studied in human liver microsomes. Biphasic kinetics was observed from the Eadie-Hofstee plot for the formation of both metabolites of M-l. For the kinetics of pentoxifylline, mean values of $V_{max1}{\;}and{\;}V_{max2}$ were 1,648 and 5,622 nmol/min/mg protein, and the estimated values of $K_{ml}{\;}and{\;}K_{m2}$ were 0.180 and 4.829 mM, respectively. For M-3, mean values of $V_{max1}{\;}and{\;}V_{max2}$ were 0.062 and 0.491 nmol/min/mg protein, and estimated values of $K_{ml}{\;}and{\;}K_{m2}$ were 0.025 and 1.216 mM. The formations of pentoxifylline and M-3 from M-1 were indentified by using several selective inhibitors of cytochrome P450 isoformes at 0.05-5 mM concentration of M-1 in human liver microsomes. For the analysis of low (0.05 mM) concentration of M-1, where the affinity was expected as low, indicated that CYPlA2 and CYP3A4 were major P450 isoforms responsible for pentoxifylline and M-3 formation. CYP3A4 and CYP2A6 appeared to be P450 isoforms responsible for M-3 formation at high (5 mM) concentration of M-1.

• 한국환경성돌연변이발암원학회지
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• 제26권3호
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• pp.86-88
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• 2006

Cytochrome P450 1A2 (CYP1A2) is a xenobiotic metabolizing enzyme that is tissue-specifically expressed in the mammalian liver. In this study, the extent of CYP1A2 promoter methylation was analyzed to determine its potential role in the regulation of CYP1A2 in diethylnitrosamine (DEN)-induced mouse liver tumors. CYP1A2 mRNA was under-expressed about three fold in DEN-induced liver tumors compared to age-matched control livers. The CYP1A2 promoter was hypermethylated in DEN-induced liver tumors compared to controls, especially in a promoter domain close to the coding region. These results suggest that promoter methylation is involved in the regulation of CYP1A2 in mouse liver tumors.

• 융합정보논문지
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• 제10권5호
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• pp.134-142
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• 2020

흡연자들의 흡연 물질 대사효소의 유전적 다형성에 따른 폐기능의 차이를 보기 위하여 질병력과 정신과적 병력이 없는 신체적·정신적으로 건강한 만 20~27세 이하의 흡연자 31명( 남 29, 여 3)을 대상으로 연구를 진행하였다. 폐활량 측정기(Wright Respirometer, Ferraris Development and Engineering Co, Ltd, UK)를 이용하여, 노력성 폐활량(Forced vital capacity, FVC), 1초간 노력성 호기량(Forced expiratory volume at one second, FEV 1), 1초간 노력성 호기량의 노력성 폐활량에 대한 비(FEV1 % FVC)을 측정하였으며, 유전자 검사는 DNA로 PCR하여 CYP1A1과 TP53의 유전자 발현검사를 하였다. 실험결과 유전자 돌연변이형이 없는 TT와 Arg/Arg의 폐기능 평균값이 가장 높았으며, CYP1A1와 lung functions의 ANOVA 분석에서 FVC의 P-값이 0.049로 그룹 간의 차이가 있는 것으로 나타났다. 즉 담배성분의 대사 활성화와 연관이 많은 Cytochrome P-450 1A1 (CYP1A1) 유전자의 돌연변이형이 없을때 FVC의 값이 높게 나타난 것이다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.301.2-301.2
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• 2002

The present study was done to determine the effect of trolox C. a hydrophilic analogue of vitamin E. on alteration in cytochrome P-450 (CYP)-dependent drug metabolism during ischemia and reperfusion. Rats were subjected to 60 min of hepatic ischemia and 5 h of reperfusion. Rats were treated intravenously with trolox C (2.5 mg/kg) or vehicle (PBS. pH 7.4), 5 min before reperfusion. Serum alanine aminotransferase and lipid peroxidation levels were markedly increased after ischemia and reperfusion. This increase was significantly suppressed by trolox C. (omitted)

• 환경생물
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• 제21권2호
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• pp.170-176
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• 2003

내분비계 장애물질이 해양생물에게 미치는 영향을 조사하기 위한 연구의 일환으로 우리나라 연안떼서 보편적으로 분포하는 p,p'-DDT, 2, 3, 7, 8-TCDD및 PCB-153을 대상으로 이들 화합물이 동해안산 명주조개의 미크로좀 중 약물대사 효소계에 미치는 형향을 살펴보았다. 명주조개의 중장선으로 만든 미크로좀에 상기한 화합물을 DMSO에 녹여 일정한 농토가 되도록 첨가하고 p,p'-DDT (0.1, 0.4 및 1.0 mM)는 30분까지 그리고 2, 3, 7, 8-TCDD (0.01, 0.04 및 0.1 ppb) 및 PCB-153 (0.01, 0.04및 0.1ppb)은 7시간까지 in vitro로 배양하면서 CYP 함량과 EROD 활성의 변화를 경시적으로 측정하였다. 그리고 용제인 DMSO만을 첨가한 미크로좀으로 sham구를 설정하였다. DDT와 배양한 경우, 0.4 및 1.0mM 배양구의 CYP농도는 노출 전에 비해 10분 후에 각각 115%와 117%로 높아졌고, EROD활성도 120%로 증가하였다. TCDD와 배양한 경우, CYP농도는 노출 전에 비해 9시간 후에 최대(103∼110%)가 되었고, BROD활성도 2시간 후에 최대(121∼139%)가 되었다. PCB-153와 배양한 경우, 0.01,0.04 및 0.10 ppb 배양구는 CYP농도가 9시간 후에 각각 107%, 112%및 112%로 최대가 되었고, EROD활성도 각각 129%,140% 및 135%로 최대가 되었다. 이것으로 세 화합물은 모두 중장선의 미크로좀에서 CYP와 ERDD수준을 높이는 유도효과가 있음을 확인할 수 있었다.

• Toxicological Research
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• 제13권1_2호
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• pp.117-125
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• 1997

In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$-aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43$\pm$0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32$\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1207-1213
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• 2020

Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus flavus (A. flavus). AFB1 is reported to have high thermal stability and is not decomposed by heat treatment during food processing. Therefore, in this study, knowing that AFB1 is metabolized by cytochrome P450 (CYP), our aim was to develop a method to detoxify A. flavus-contaminated maize, under normal temperature and pressure, using Escherichia coli expressing human CYP3A4. First, the metabolic activity of AFB1 by recombinant human CYP3A4 was evaluated. As a result, we confirmed that recombinant human CYP3A4 metabolizes 98% of AFB1. Next, we found that aflatoxin Q1, a metabolite of AFB1 was no longer mutagenic. Furthermore, we revealed that about 50% of the AFB1 metabolic activity can be maintained for 3 months when E. coli expressing human CYP3A4 is freeze-dried in the presence of trehalose. Finally, we found that 80% of AFB1 in A. flavus-contaminated maize was metabolized by E. coli expressing human CYP3A4 in the presence of surfactant triton X-405 at a final concentration of 10% (v/v). From these results, we conclude that AFB1 in A. flavus-contaminated maize can be detoxified under normal temperature and pressure by using E. coli expressing human CYP3A4.