• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.295-298
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• 2006

Actinomycetes are ubiquitous Gram-positive soil bacteria and a group of the most important industrial microorganisms for the biosynthesis of many valuable secondary metabolites as well as the source of various bioconversion enzymes. Cytochrome P450 hydroxylase (CYP), a hemebinding protein, is known to be involved in the modification of various natural compounds, including polyketides, fatty acids, steroids, and some aromatic compounds. Previously, six different novel CYP genes were isolated from a rare actinomycetes called Sebekia benihana, and they were completely sequenced, revealing significant amino acid similarities to previously known CYP genes involved in Streptomyces secondary metabolism. In the present study, these six CYP genes were functionally expressed in Streptomyces lividans, using an $ermE^{*}$ promoter-containing Streptomyces expression vector. Among six CYP genes, two S. benihana CYP genes (CYP503 and CYP504) showed strong hydroxylation activities toward 7-ethoxycoumarin. Furthermore, the recombinant S. lividans containing both the S. benihana CYP506-ferredoxin genes as well as the S. coelicolor feredoxin reductase gene also demonstrated cyclosporin A hydroxylation activity, suggesting potential application of actinomycetes CYPs for the biocatalysts of natural product bioconversion.

• Development and Reproduction
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• v.10 no.2
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• pp.127-133
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• 2006

[ $17\;{\alpha}-hydroxylase/17,20-lyase(P450_{C17})$ ] is the key enzyme mediating the conversion of progesterone to $17\;{\alpha}-hydroxyprogesterone$, ultimately to androstenedione during steroidogenesis. R. dybowskii's ovarian $P450_{C17}$ cDNA was cloned to understand the regulatory mechanism of ovarian steroidogenic pathway at the molecular level in amphibian. A 2.5kb cDNA clone encoding a single open-reading frame with a 519 deduced amino acid was isolated with the screening of ovarian cDNA library. This sequence contained the three highly conserved domains as seen in $P450_{C17}$ of other species. The comparison of amino acid sequence of Rana $P450_{C17}$ with other animal's $P450_{C17}$ showed relatively high identity with 76% in Xenopus, 63% in chicken, 60% in rainbow trout, and 45% in human. Phylogenic analysis also indicated that Rana $P450_{C17}$ gene was evolutionary well conserved among vertebrate. Northern analysis indicated that the two different sizes of $P450_{C17}$ transcripts with approximately 2.5 and 3.6kb were detected in ovary tissue, but not in other tissues. The expression vector of Rana $P450_{C17}$ clearly showed the $17\;{\alpha}-hydroxylase$ activity converting the exogenous progesterone into $17\;{\alpha}-hydroxyprogesterone$ in the nonsteroidogenic COS-1 cells. Therefore, Rana $P450_{C17}$ cDNA is very useful to investigate the molecular mechanism of the ovarian steroidogenesis in amphibian.

• BMB Reports
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• v.31 no.4
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• pp.391-398
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• 1998

In vitro effects of acetone, methanol, and dimethylsulfoxide (DMSO) on liver microsomal cytochrome P450 (P450) content, and P450-dependent arylhydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were studied in rats. Acetone at 1% (v/v) enhanced the content ofP450, assayed spectrally in 3-methylcholanethrene (MC)- and ${\beta}-naphthoflavone$ (BNF)-inducible microsomes by 18 and 7%, respectively. Methanol, up to 5% (v/v) applied, also showed enhancement effects on P450 content in liver microsomes from rats treated with phenobarbital (PB), MC, and BNF, as well as uninduced microsomes with similar but low strength. DMSO, however, did not show such enhancing effects at the ranges of the concentrations applied. AHH and ECOD activities in MC-inducible microsomes were also enhanced by acetone at 1%, which was in proportion to the increase in P450 content by the same concentration. However, the P450 content, and AHH and ECOD activities, were decreased by increasing the concentration of acetone. Methanol at the same concentration with acetone also enhanced ECOD activity but not AHH activity in MCinducible microsomes. The enhancing effect of acetone on the enzymes was negligible when the microsomes were pretreated with a specific monoclonal antibody of MC-inducible isozyme. The difference in the effects of these solvents on P450 system might be due to their different properties that cause the P450 active site to be exposed in milieu.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.17-28
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• 1998

Drug-metabolizing activities of Korean native cattle and swine were investigated from viewpoints of the cytochrome P-450's level, their dependent mixed function oxidase activities, the reactive oxygen species formation and cytosolic enzyme acitivities from each liver homogenates. Level of cytochrome P-450 in the liver microsome of Korean native cattle was $0.28{\pm}0.05nmole/mg$ and that in pigs $0.35{\pm}0.03nmole/mg$. Level of cytochrome $b_5$ of Korean native cattle was $0.24{\pm}0.06nmole/mg$, and that of pigs $0.2{\pm}0.05nmole/mg$, showing no difference between two species. NADPH P-450 reductase were higher in Korean native cattle ($58.3{\pm}5.3nmole/mg/min$) than in pigs ($29.9{\pm}3.8nmole/mg/min$)(p<0.01). The activities of cytochrome P-450 dependent monooxygenases such as ethoxyresorufin O-deethylase (cattle, $96.5{\pm}12.5nmole/mg/min$ ; pigs, $13.6{\pm}2.1nmole/mg/min$), N-benzphetamine N-demethylase (cattle, $5.23{\pm}0.82nmole/mg/min$ ; pigs, $0.76{\pm}0.3nmole/mg/min$) and aniline hydroxylase (cattle, $0.95{\pm}0.1nmole/mg/min$ ; pigs, $0.33{\pm}0.08nmole/mg/min$) were much higher in Korean native cattle than in swine(p<0.01). However, the activity of testosterone $7{\alpha}$-hydroxylase was higher in swine ($90.4{\pm}1.2nmole/mg/min$) than cattle (cattle, $32.8{\pm}1.2nmole/mg/min$). Interestingly, testosterone $16{\alpha}$-hydroxylase, a marker enzyme for P-450 IIA was not detected in both animal species. These results suggest that Korean native cattle and pigs have high contents of P-450 IA1 and P-450 IIIA. Total sulfhydryl compound (cattle, $10.3{\pm}1.1nmole/mg$ ; Pigs, $14.5{\pm}1.8nmole/mg$) and glutathione related enzymes except glutathione reductase (cattle, $38.1{\pm}7.9nmole/mg/min$; swine, $22{\pm}3.6nmole/mg/min$) showed higher levels in swine than in Korean native cattle. Superoxide dismutase (cattle, $7.64{\pm}0.84nmole/mg/min$ ; pigs, $4.47{\pm}0.94nmole/mg/min$) and catalase (cattle, $30.4{\pm}3.7nmole/mg/min$ ; pigs, $17.2{\pm}1.8nmole/mg/min$) were remarkably higher in Korean native cattle than in swine (p<0.05).

• BMB Reports
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• v.30 no.4
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• pp.237-239
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• 1997

The effects of trichloroethylene (TRI) on the induction of cytochrome P-450 (CYP) and several other related enzymes in Sprague Dawley rats were investigated Rats were treated with TRI 150. 300. 600 mg/kg body weight in corn oil intra peritoneally once a day for 2 days. The total contents of microsomal CYP and cytochrome $b_5\;(b_5)$ decreased with the increase of TRI concentration. but the activity of p-nitrophenol hydroxylase increased with the increase of TRI dosage (p<0.05). Western blot analysis which utilized monoclonal antibodies against CYP2E1 also showed a significant increase in the CYP2E band density. The increase of the activity of pentoxyresolufin-O-deethylase also was observed with the TRI treatment (p<0.05) although there was no significant increase in the cytochrome CYP2B1/2 in Western blotting The TRI did not affect the induction of aryl hydrocarbon hydroxylase. These findings suggest that the CYP2E1 is the primary enzyme which could be induced by TRI treatment in rats.

• Journal of Life Science
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• v.19 no.8
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• pp.1039-1046
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• 2009

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

• BMB Reports
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• v.35 no.6
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• pp.615-622
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• 2002

The purpose of this study was to determine the effects of dietary garlic powder on diethylnitrosamine (DEN)-induced hepatocarcinogenesis and cytochrome P450 (CYP) enzymes in weaning male Sprague-Dawley rats by using the medium-term bioassay system of Ito et al. The rats were fed diets that contained 0, 0.5, 2.0 or 5.0% garlic powder for 8 weeks, beginning the diets with the intraperitoneal (i.p.) injection of DEN. The areas of placental glutathione S-transferase (GST-P) positive foci, an effective marker for DEN-initiated lesions, were significantly decreased in the rats that were fed garlic-powder diets; the numbers were significantly decreased only in the 2.0 and 5.0% garlic-powder diets. The p-nitrophenol hydroxylase (PNPH) activities and protein levels of CYP 2E1 in the hepatic microsomes of the rats that were fed the 2.0 and 5.0% garlic powder diet were much lower than those of the basal-diet groups. Pentoxyresorufin O-dealkylase (PROD) activity and CYP 2B1 protein level were not influenced by the garlic-powder diets and carcinogen treatment. Therefore, the suppression of CYP 2E1 by garlic in the diet might influence the formation of preneoplastic foci during hepatocarcinogenesis in rats that are initiated with DEN.

• KSBB Journal
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• v.19 no.4
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• pp.308-314
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• 2004

A degenerate set of PCR primers based on two conserved regions (heme binding region and oxygen ligand pocket) were designed and successfully applied to amplify DNA fragments of cytochrome P450 hydroxylase (CYP) genes from a rare actinomycetes, S. benihana. The PCR amplified products were employed as a DNA probe to clone the entire CYP genes from S. benihana genomic library. Five different CYP-positive cosmids were isolated by colony hybridization as well as PCR confirmation. The complete nucleotide sequencing of five different CYP genes revealed that each unique CYP showed a significant amino acid homology to previously-known CYP genes involved in streptomycetes secondary metabolism. In addition, four CYP genes (CYP502, CYP503, CYP504, CYP506) were found to be linked to ferredoxin genes in the chromosome, and the CYP503 gene showed the high degree of amino acid similarity to the previously well-characterized CYP105 family in streptomycetes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1992.05a
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• pp.54-54
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• 1992

약물, hormone, 독성물질등의 대사능과 발암 가능성등이 간장 장해시 및 ketosis시에 달라지는 원인과 기전, 독성물질 대사효소의 변동과 그 작용기전을 규명하고자, 대표적인 간장장해 물질인 carbon tetrachloride를 rat에 투여하여 간장 장해를 일으키고, 당뇨병, starvation, high-fat diet처리하여 ketosls상태를 만든 후에, specific cytochrome P45O polyclonal antibodies와 cDNA probes를 사용하여, enzyme activitieg, Western immunoblot analysis와 mRNA Northern blot analysis 등을 실험하여, 간장 장해와 ketosis시 cytochrome P45O의 변동과 그 작용기전, regulation을 규명하고자 하였다. 실험 결과, $CCl_4$투여후 P450IIE enzyme (aniline hydroxylase) 활성이 시간 의존적으로 급격히 떨어졌고, P450IIE protein양이 똑같은 방식으로 감소되었으나 mRNA level은 변화가 없었다. $CCl_4$에 의해서 P450IIE는 protein의 특이적인 파괴에 의한 post-translational reduction됨을 알 수 있었다. 반면에 당뇨병, starvation, high-fat diet등 ketosis시에는 P450IIE 효소활성이 2-3배 증가되었고, P450IIE protein양도 같은 수준으로 증가되었으며, mRNA도 증가 되었다. Ketosis시에는 P450IIE가 pretranslational activation됨을 알 수 있었다.

• Environmental Analysis Health and Toxicology
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• v.7 no.3_4
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• pp.17-35
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• 1992

In this study, it was examined the toxic effects of combination of buprofezin and carbaryl on hematological, biological and enzymetic parameters in rats. The administration of buprofezin or carbaryl both induced the tissue content of cytochrome P-450 and furthermore, the combination of the both increased significantly the liver content of cytochrome P-450 in rat. But cytochrome P-450 and NADPH -cytochrome c reductase activities in kidney were slightly increased. Administration of carbaryl and combination of the both also significantly increased hepatic aniline hydroxylase activity. In addition, in the combination group, glucose-6-phosphatase and lipid peroxidase activities were changed in the rat liver. Furthermore, cholinesterase was inhibited in rats treated with carbaryl or the combination of buprofezin and carbaryl. The above results suggested that the combined administration of buprofezin and carbaryl can induce more toxic effects than the single administration of buprofezin or carbaryl.