• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.44-54
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• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.356-365
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• 2004

In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.

• 환경생물
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• 제26권2호
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• pp.87-93
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• 2008

낙동강 상류로부터 유입되는 각종 오염물질로 심하게 오염된 낙동강 하구역에서 오염정도가 다른 두 지역으로부터 문절망둑 Acanthogobius flavimanus을 채집하여 이들의 간중량지수(HSI), 생식선중량지수(GSI), 해독효소계 및 성 호르몬 수준을 비교하였다. 해독효소계로는 cytochrome P450 (CYP), NADPH-cytochrome P450 reductase (P450R), NADH-cytochrome b5 reductase (b5R), ethokyresorufin deethylase (EROD), glutathione S-transferase (GST)를 조사하였고, 성 호르몬으로는 자성호르몬인 17$\beta$-estradio(E2)을 비롯하여 웅성호르몬인 testosterone (TT), 11-ketotestolterone (11-KT)을 측정하였다. 그 결과, HSI는 site 1에서 잡은 것이 암수 모두 유의적으로 컸고, GSI는 site 1의 암컷에서 유의적으로 작았다. 그리고 성호르몬 중 11-KT과 TT농도는 오염지역에 따라 차이를 보이지 않았지만, E2농도는 site 1에서 유의적으로 높았다(p<0.05).그리고 해독효소계의 수준은 site 1의 것이 CYP와 EROD수준은 유의적으로 낮았던 반면에 P450R, b5R 및 GST 활성은 site 1에서 높았다. 이들 결과를 정리하면, 낙동강 하구에 서식하는 문절망둑은 성 호르몬 대사를 교란시키는 화합물에 의해 영향을 받고 있으며, 특히 암컷이 더욱 크게 받는다는 것을 확인할 수 있었다.

• ALGAE
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• 제30권3호
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• pp.233-239
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• 2015

Microalgae research is gaining momentum because of their potential biotechnological applications, including the generation of biofuels. Genome sequencing analysis of two model microalgal species, polar free-living Coccomyxa sp. C-169 and symbiotic Chlorella sp. NC64A, revealed insights into the factors responsible for their lifestyle and unravelled biotechnologically valuable proteins. However, genome sequence analysis under-explored cytochrome P450 monooxygenases (P450s), heme-thiolate proteins ubiquitously present in species belonging to different biological kingdoms. In this study we performed genome data-mining, annotation and comparative analysis of P450s in these two model algal species. Sixty-nine P450s were found in two algal species. Coccomyxa sp. showed 40 P450s and Chlorella sp. showed 29 P450s in their genome. Sixty-eight P450s (>100 amino acid in length) were grouped into 32 P450 families and 46 P450 subfamilies. Among the P450 families, 27 P450 families were novel and not found in other biological kingdoms. The new P450 families are CYP745-CYP747, CYP845-CYP863, and CYP904-CYP908. Five P450 families, CYP51, CYP97, CYP710, CYP745, and CYP746, were commonly found between two algal species and 16 and 11 P450 families were unique to Coccomyxa sp. and Chlorella sp. Synteny analysis and gene-structure analysis revealed P450 duplications in both species. Functional analysis based on homolog P450s suggested that CYP51 and CYP710 family members are involved in membrane ergosterol biosynthesis. CYP55 and CYP97 family members are involved in nitric oxide reduction and biosynthesis of carotenoids. This is the first report on comparative analysis of P450s in the microalgal species Coccomyxa sp. C-169 and Chlorella sp. NC64A.

• Toxicological Research
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• 제26권1호
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• pp.47-52
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• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

• 한국연초학회지
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• 제20권1호
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• pp.99-107
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• 1998

Numerous studies have demonstrated a negative association between cigarette smoking and Parkinson's disease. The present study was undertaken to investigate whether chronic exposure of mice to cigarette smoke a(footed the metabolism of 1-methyl-1113,6-tetrahydro-pyridine (MPTP) by cytochrome P4SO (P-450) or flavin-containing monooxygenase (FMO) in the hepatic microsomes of C57BL6/J mice. Adult male C57BL6/J mice were exposed to mainstream smoke generated from 15 cigarettes for 10 min a day and 5 day per week for 6 weeks. MPTP (10 mg/kg body weight) was administered to mice by subcutaneous injection for 6 consecutive days. Microsolnal P-450 content was increased by MPTP, smoke exposure, or both, but NADPH cytochrome P-450 reductase activity was rather decreased by the same treatments. The activities of benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase were significantly increased by the exposure of cigarette smoke, but were not or little affected by MPTP treatment. Benzphetamine N-demethylase activity was not affected either by MPTP treatment or by cigarette smoke exposure, but it was significantly increased by the combined MPTP treatment with cigarette smoke exposure, showing their synergic effect for the induction of the enzyme activity. Interestingly, in vitro studies of hepatic FMO and P-450 system both O-oxygenation and N-demethylation of MPTP were increased in the smoke-exposed or in the MPTP-treated mice. These results suggest that the enhancement in the N-demethylation as well as O-deethylation of P-450 system and in the N-oxygenation of FMO activity by cigarette smoke exposure in mouse liver may contribute to attenuating the neurotoxic effects of MPTP on the nigrostriatal dopaminergic neurons.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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• pp.777-784
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• 2020

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named 'CYP102A15 and CYP102A170,' from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724,respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C₁₂-C₂₀, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with K_m 15.3 μM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 μM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.107-107
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• 1997

Cytochrome P450 enzymes have been intensively investigated in hepatic tissues and several mammalian cell lines. Compared to most studies about cytochrome P450 isozymes in liver in vivo and hepatic, cell lines in vitro, the study of cytochrome P450IA1 in human breast cancer cells could be very important to understand the mechanism of the regulation of CYPIA1 gene expression and cell growth. MCF-7 human breast cancer cells are well characterized to study estrogen and antiestrogen action due to the fact that they contain high level of estrogen receptor and have biological markers characterized. And also MCF-7 cells express high level of arylhydrocarbon hydroxylase activity and human cytochrome P450IA1 cDNA was cloned from MCF-7 cells. Ah receptor was characterized in many breast cancer cell lines and polycyclic aromatic hydrocarbon such as 3-MC induced the expression of CYPIA1 gene and cytochrome P450- dependent monooxygenase activity. We undertook a study to examine the effect of estrogens and other chemicals on the regulation of human CYPIA1 gene expression in MCF-7 cells via RTPCR analysis, that might help us to understand the mechanism of the regulation of CYPIA1 gene expression and MCF-7 cell growth. Expression vector containing the functional 5'-regulatory region of human CYPIA1 fused to the CAT reporter gene was transfected into estrogen receptor positive MCF-T cells or estrogen receptor negative MDA-MB-231 cells. After these cells were treated with various chemicals, RTPCR was carried out to measure both CYPIA1 mRNA and CAT mRNA levels. 1nM 3-MC increased in both P450 and CAT mRNA levels over those of control by two folds in MCF-7 cells but does not in MDA-MB-231 cells. Estrogen or tamoxifen or retinoic acid or chrysin decreased in both P450 and CAT mRNA levels that were induced by 3-MC in MCF-7 when each chemical was administered with 3-MC concomitantly. These results suggested that the level of CYPIA1 gene expression is modulated with estrogen-related molecules and make it possible to speculate that ER is related to CYPIA1 gene expression and cell growth in breast cancer cells. [Supported by grants from the Korean Ministry of Education ]

• Toxicological Research
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• 제3권1호
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• pp.1-8
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• 1987

In vitro induction of cytochrome 450 associated monooxygenase activities by phenobarbital (PB) and 3-methylcholanthrene (MC) was investigated in primary cultures of adult rat hepatocytes. PB and MC were added to the culture 24 hr after the initial plating of hepatocytes. A signiftcant increase of the activities of 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase were observed in MC and PB treated culture. MC caused about 500% induction of the initial oxidation rates of both enzymes in 48 hr. However the PB maintained both enzyme activities close to the level of freshly isolated hepatocytes. Biphenyl 4-hydroxylase and aminopyrine N-demethylase activities were also induced by MC and PB. But the level of induction was less than that occuring with 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase. When aflatoxin $B_1$ was added to the hepatocyte cultures which have been treated with MC or PB, it caused a significant increase of the unscheduled DNA synthesis at higher dose of aflatoxin $B_1$ as compared to those of untreated control hepatocyte cultures. The results suggest that microsomal enzyme activities can be selectively controlled preferably in hepatocyte cultures by the in vitro induction method. This principle may be useful for studying the metabolism and other toxicological studies.

• Biomolecules & Therapeutics
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• 제20권6호
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• pp.562-568
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• 2012

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics ($k_{cat}=4120\;min^{-1}$, $K_m=77{\mu}M$ for MTT and $k_{cat}=6580\;min^{-1}$, $K_m=51{\mu}M$ for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.