• Journal of Preventive Medicine and Public Health
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• v.28 no.1 s.49
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• pp.141-152
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• 1995

This study was performed to find out the influences of ethanol on the metabolism of trichloroethylene(TRI) in rats. TRI in corn oil at the dosage of 150, 300, 600 mg/kg was injected peritoneally once a day for two days to two groups. In one group ethanol(4 g/kg) was taken orally 30 minutes before TRI injection, and the other group ethanol was not. The results of experiments are as follows: 1. The contents of cytochrome P-450 and $b_5$ had inverse relationship with in-jected TRI amounts in both groups. 2. The activity of NADPH P-450 reductase was decreased slowly in TRI injected group related with TRI amount, but decreased drastically in the group pretreated with ethanol. 3. The activity of NADH $b_5$ reductase had relationship with injected nt amount , but the statistical significance was found only in the groups of 300 and 600 mg/kg of TRI injected without relevance to ethanol when compared with the group that was not injected. 4. The activity of ADH was more decreased and ALDH activity was more increased in groups that TRI injected and ethanol was pretreated with ethanol groups than in group without any treatment. These results suggest that ethanol may inhibit epoxide formulation, the first step of TRI metabolism, and change from TCE-OH to TCA also.

• Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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• 2003.04a
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• pp.188-191
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• 2003

Sing]e-well-push-pull tests were developed for use in assessing the feasibility of in-situ aerobic cometabolism of chlorinated aliphatic hydrocarbons (CAHs). The series includes Transport tests, Biostimulation tests, and Activity tests. Transport tests are conducted to evaluate the mobility of solutes used in subsequent tests. These included bromide or chloride (conservative tracers), propane (growth substrate), ethylene, propylene (CAH surrogates), dissolved oxygen (electron acceptor) and nitrate (a minor nutrient). Tests were conducted at an experimental well field of Oregon State University. At this site, extraction phase breakthrough curves for all solutes were similar, indicating apparent conservative transport of the dissolved gases and nitrate prior to biostimulation. Biostimulation tests were conducted to stimulate propane-utilizing activity of indigenous microorganisms and consisted of sequential injections of site groundwater containing dissolved propane and oxygen. Biostimulation was detected by the increase in rates of propane and oxygen utilization after each injection. Activity tests were conducted to quantify rates of substrate utilization and to confirm that CAH-transforming activity had been stimulated. In particular, the transformation of injected CAH surrogates ethylene and propylene to the cometabolic byproducts ethylene oxide and propylene oxide provided evidence that activity of the monooxygenase enzyme system, responsible for aerobic cometabolic transformations of CAHs had been stimulated. Estimated zero-order transformation rates decreased in the order propane > ethylene > propylene. The series of push-pu3l tests developed and field tested in this study should prove useful for conducting rapid, low-cost feasibility assessments for in situ aerobic cometabolism of CAHs.

• Biomolecules & Therapeutics
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• v.20 no.6
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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.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.187-195
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• 2000

Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) of Pseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 genes. All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A). However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream of pcbC gene. Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. The orf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues. A deduced amino acid sequence of the orf1 gene product exhibited 21-33％ identity with those of indole dioxygenase and phenol hydroxylase components. The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. The orf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues. A deduced amino acid sequence of the orf2 gene product exhibited 31％ identity with that of a nitrilotriacetate monooxygenase component.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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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.

• KSBB Journal
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• v.8 no.4
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• pp.341-350
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• 1993

As an effort to develop an alternative transportation fuel, the production of methanol from methane gas was studied using the resting cells of an obligatory methanotroph, Methylosinus trichosporium OB3b. The reaction was carried out in high concentration phosphate buffer solutions with the flask-grown cells containing the exclusively cytoplasmic methane monooxygenase (sMMO) activity. The methanol accumulation rate was observed to be 79nmo1/mg·min during the initial 4.5hr. Phosphate-dependent inhibition was found for both sMMO and methanol dehydrogenase (MDH) activities, and the inhibition constants were 185mM and 42mM, respectively. The inhibition mode was noncompetitive. Methanol was found to be very inhibitory to the sMMO activity and the inhibition constant (noncompetitive) was 21mM when propylene was used as substrate. The sMO activity in the resting cells was declined very fast and the rate became very high during the methanol production. These results indicate that the use of M. trichosporium OB3b as a biocatalyst for the methanol production is heavily dependent on the stable maintenance of the whole-cell SMO activity as well as the effective alleviation of product inhibition.

• KSBB Journal
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• v.11 no.6
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• pp.642-648
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• 1996

The effects of EDTA(Ethylene diamine tetraacetic acid), Cu, temperature, and gas(methane and oxygen) composition on methanol production from methane with Methylosinus trichosporium were investigated. In this experiment EDTA was found to be a potential methanol dehydrogenase inhibitor since it causes methanol accumulation and 6mM was found to be optimum concentration of EDTA for methanol production. When Cu was added in culture media, the produced methanol concentration level was increased. Hence it is believed that Cu enhanced the particulate methane monooxygenase formation and consequently the addition of Cu could increase the methanol production from methane. In this experiment the optimum concentration of Cu was found to be 1mM for methanol production. When temperature was shifted down from $30^{\circ}C to 25^{\circ}C$, the methanol production level was enhanced by 50%. When the ratio of methane to oxygen in gas phase was increased to 2.3 from 1, produced methanol concentration was also enhanced by 100%.

• Journal of Life Science
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• v.13 no.6
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• pp.970-975
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• 2003

Microbial trichloroethylene (TCE) degradation using trickling biofilter (TBF) is a cost-effective treatment method, in which monooxygenase (MO) fortuitously transforms TCE via cometabolism. Simple TBF, however, could not be stably operated for long-term treatment of TCE due to the contradictory characteristics of cometabolism. In this paper, microbial biocatalyst and biofilm reactor system, a two-stage continuous stirred tank reactor (CSTR)/TBF system using Burkholderia cepacia G4 and Methylosinus trichosporium OB3b, are evaluated for the long-term continuous treatment of TCE. The maximum TCE elimination capacities were in the range of 28 and 525 mg TCE/1$.$day. The reactor systems were stably operated for more than 3∼12 months.

• Biomolecules & Therapeutics
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• v.17 no.2
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• pp.156-161
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• 2009

The human cytochrome P450 4A11 is the major monooxygenase to oxidize the fatty acids and arachidonic acid. The production of 20-hydroxyeicosatetraenoic acid by P450 4A11 has been implicated in the regulation of vascular tone and blood pressure. Oxidation reaction by P450 4A11 requires its reduction partners, NADPH-P450 reductase (NPR). We report the functional expression in Escherichia coli of bicistronic constructs consisting of P450 4A11 encoded by the first cistron and the electron donor protein, NPR by the second. Typical P450 expression levels of wild type and several N-terminal modified mutants was observed in culture media and prepared membrane fractions. The expression of functional NPR in the constructed P450 4A11: NPR bicistronic system was clearly verified by reduction of nitroblue tetrazolium. Membrane preparation containing P450 4A11 and NPR efficiently oxidized lauric acid mainly to $\omega$-hydroxylauric acid. Bicistronic coexpression of P450 4A11 and NPR in E. coli cells can be extended toward identification of novel drug metabolites or therapeutic agents involved in P450 4A11 dependent signal pathways.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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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.