Molecular Cloning and Expression of Fusion Proteins Containing Human Cytochrome P450 3As and Rat NADPH-P450 Reductase in Escherichia coli

Chun, Young-Jin,Guengerich, F-Peter Korean Society of ToxicologyKorea Environmental Mu 2002 Toxicological Research Vol.18 No.3

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

Kinetic deuterium isotope effects for 7-alkoxycoumarin <i>O</i>-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes

Kim, Keon-Hee,Isin, Emre M.,Yun, Chul-Ho,Kim, Dong-Hyun,Guengerich, F. BLACKWELL 2006 FEBS JOURNAL Vol.273 No.10

<P>7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin <I>O</I>-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for <I>O</I>-demethylation, 6.1 for <I>O</I>-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for <I>O</I>-demethylation, 6.1 for <I>O</I>-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin <I>O</I>-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3–8.1 for <I>O</I>-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.</P>

Sex- and Tissue-related Expression of Two Types of P450 Aromatase mRNA in the Protandrous Black Porgy, Acanthopagrus schlegeli, during Sex Reversal: Expression Profiles Following Exogenous Hormone Administration

Tae Sun Min,Kwang Wook An,길경석,최철영 한국통합생물학회 2009 Animal cells and systems Vol.13 No.4

Cytochrome P450 aromatase (P450arom) catalyzes the conversion of androgens to estrogens and plays an important role in reproduction and development in vertebrates. We investigated the expression patterns of ovarian P450arom (P450aromA) and brain P450arom (P450aromB) mRNA during sex change in black porgy. Maturity was divided into seven stages from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). P450aromA expression was significantly higher in the ovarian portion of mostly-ovarian stage fish, and P450aromB expression was highest in the brain of black porgy with mostly-ovarian gonads. Histology showed that testicular tissues were disintegrated with the development of ovarian tissue associated with an increase in the expression of the two P450arom mRNAs during sex change. Interestingly, among various tissues, P450aromA was only expressed in the ovary, and P450aromB was only expressed in the brain. To understand the role of gonadotropin-releasing hormone (GnRH) and estradiol (E2), we injected exogenous hormone (GnRH analogue [GnRHa] and E2) into immature black porgy. In the GnRHa group, expression of the two P450arom genes decreased 12 h after injection, and expression of the two P450arom genes were significantly higher at 6 d after E2 injection. These results provide useful baseline knowledge on the mechanism of natural sex change in black porgy. Cytochrome P450 aromatase (P450arom) catalyzes the conversion of androgens to estrogens and plays an important role in reproduction and development in vertebrates. We investigated the expression patterns of ovarian P450arom (P450aromA) and brain P450arom (P450aromB) mRNA during sex change in black porgy. Maturity was divided into seven stages from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). P450aromA expression was significantly higher in the ovarian portion of mostly-ovarian stage fish, and P450aromB expression was highest in the brain of black porgy with mostly-ovarian gonads. Histology showed that testicular tissues were disintegrated with the development of ovarian tissue associated with an increase in the expression of the two P450arom mRNAs during sex change. Interestingly, among various tissues, P450aromA was only expressed in the ovary, and P450aromB was only expressed in the brain. To understand the role of gonadotropin-releasing hormone (GnRH) and estradiol (E2), we injected exogenous hormone (GnRH analogue [GnRHa] and E2) into immature black porgy. In the GnRHa group, expression of the two P450arom genes decreased 12 h after injection, and expression of the two P450arom genes were significantly higher at 6 d after E2 injection. These results provide useful baseline knowledge on the mechanism of natural sex change in black porgy.

Functional Characterization of Pharmcogenetic Variants of Human Cytochrome P450 2C9 in Korean Populations

( Myung-a Cho ),( Jihoon G Yoon ),( Vitchan Kim ),( Harim Kim ),( Rowoon Lee ),( Min Goo Lee ),( Donghak Kim ) 한국응용약물학회 2019 Biomolecules & Therapeutics(구 응용약물학회지) Vol.27 No.6

Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants―including three novel variants F69S, L310V, and Q324X―that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high k_cat values; however, their K_m values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher K_m and lower k_cat values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower k_cat and K_m values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.

Cytochrome P450 Networks in Chemical Space

Lee, So-Young,Kim, Dong-Sup 대한약학회 2010 Archives of Pharmacal Research Vol.33 No.9

A global analysis of the chemical space of cytochrome P450 enzymes in humans has not been achieved despite its great importance for drug metabolism and drug-drug interactions. We analyzed the global characteristics of cytochrome P450s by building several networks at the family, subfamily, and gene levels from information on P450 substrates, inducers, and inhibitors. These networks provide insight into the relationship of cytochrome P450 isoforms on the metabolism of drugs, changes in drug activity, and the promiscuous properties of each cytochrome P450 enzyme. From the networks, we analyzed the centrality of nodes and measured the strength of correlations between two nodes by drawing promiscuity maps. In addition, heat maps were generated to cluster cytochrome P450s by their similarity within three chemical spaces (substrates, inducers, and inhibitors). We observed the intra-linking and interlinking connections between three chemical spaces, the relative correlations of a given cytochrome P450 isoform with other isoforms, and the similarity of the metabolizing ability and changing pattern by chemicals. These results provide a global view of the relationship and similarity of cytochrome P450s on various chemical spaces at various levels. The measures of the strength of connection between two cytochrome P450s and the heat-map information could be used to predict drug-drug interactions, perform phylogenetic analyses, and further understand cooperative properties of these enzymes.

Cytochrome P450 monooxygenase analysis in free-living and symbiotic microalgae Coccomyxa sp. C-169 and Chlorella sp. NC64A

Ntsane Trevor Mthakathi,Ipeleng Kopano Rosinah Kgosiemang,Wanping Chen,Molikeng Eric Mohlatsane,Thebeyapelo Jacob Mojahi,Jae-Hyuk Yu,Samson Sitheni Mashele,Khajamohiddin Syed 한국조류학회I 2015 ALGAE Vol.30 No.3

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.

Cytochrome P450 monooxygenase analysis in free-living and symbiotic microalgae Coccomyxa sp. C-169 and Chlorella sp. NC64A

Mthakathi, Ntsane Trevor,Kgosiemang, Ipeleng Kopano Rosinah,Chen, Wanping,Mohlatsane, Molikeng Eric,Mojahi, Thebeyapelo Jacob,Yu, Jae-Hyuk,Mashele, Samson Sitheni,Syed, Khajamohiddin The Korean Society of Phycology 2015 ALGAE Vol.30 No.3

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.

Purification and Charactrization of Sex Specific Isozyme of P-450 from Rat Liver and Testosterone Hydroxylation Catalyzed by P-450 Isozyme

HONG, Young-Sook 이화여자대학교 생명과학연구소 1991 생명과학연구논문집 Vol.2 No.-

Three microsomal cytochrome(P-450)were purified to electrophoretic homogeneity from livers of adult male rats (P-450_M-1, P-450_M-2, and P-450_M-3). Purified P-450_M-1, P-450_M-2, and P-450_M-3 had apparent molecular weights of 51,000,48,000 and 49,000as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The carbon monoxide-difference spectra of reduced P-450_M-1, P-450_M-2, and P-450_M-3 showed an absorption maximum at 451,451 and 448 nm, respectively. The antibodies against P-450_M-2, did not crossreact with the other forms in the ouchterlony double diffusion test, whereas the immunodiffusion test showed immunocrosseactivity between P-450_M-1, and P-450_M-3 In a reconstituted system containing NADPH and NADPH -cytochrome P-450 reductase, P-450_M-1 and P-450_M-2 oxidized 7-ethoxycoumarin at a high rate, whereas the other forms had low activity toward 7-ethoxycoumarin. The two forms (P-450_M-1, P-450_M-2)showed high activity toward benzo(a) pyrene. P-450_M-1 catalyzed the hydroxylation of testosterone at the 16∂ and 2∂ position, whereas P-450_M-2, catalyzed the 15∂-hydroxylation of the same substrate. The precipitating anti-M-1 MAb inhibited by more than 90% androstenedione 16-∂-hydroxylase activity of untreated rat microsomes, with no inhibition of microsomal 6-β or 7-∂-hydraxylation was affected by the MAb.

High Yield Bacterial Expression and Purification of Active Cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis Membrane Protein

Hee Jung Yang(양희정),Wanyeon Kim(김완연),Young Ju Yun(윤영주),Ji Won Yoon(윤지원),TaeWoo Kwon(권태우),HyeSook Youn(윤혜숙),BuHyun Youn(윤부현) 한국생명과학회 2009 생명과학회지 Vol.19 No.8

다양한 천연물의 합성대사에 관여하는 식물 cytochrome P450 (P450s)은 그 기능적 다양성에도 불구하고, 이들 효소의 광범위한 기질 특이성을 설명해 줄 수 있는 구조분석에 대해서는 충분한 연구가 이루어지지 못하고 있는 실정이다. 식물 p-coumarate 3-hydroxylase (C3H)에 의해 매개되는 효소 반응은 lignin 과 다양한 phenylpropanoid 부산물들의 생합성에 매우 중요한 것으로 여겨지지만, 막 단백질인 C3H의 발현 및 정제가 효과적으로 이루어지지 못하여, 활성을 측정하기 위한 분석방법이 체계화 되지 못하고 있다. C3H의 작용기작과 기질특이성에 대해 폭넓은 이해를 위한 구조분석의 선행단계는 활성을 갖는 C3H를 밀리그램 단위로 분리, 정제하는 실험적 방법을 확립하는 것이라 할 수 있다. 이를 위해, 본 연구에서는 다양한 돌연변이 방법을 도입하여 식물 막단백질 C3H를 대장균 시스템에서 효과적으로 발현 및 정제할 수 있는 시스템을 사용하였다. 변형된 cytochrome P450 C3H (C3Hmod)을 세포막으로부터 고농도의 염완충용액을 이용하여 계면활성제 없이 추출하였으며, 2단계 chromatography를 통해 활성을 유지한 상태로 분리할 수 있었다. 이러한 실험적 기법은 NMR 및 X-ray crystallography와 같은 구조분석을 통한 C3H의 효과적인 분석에 적용될 수 있을 것이며, 또한 다른 식물 cytochrome P450 단백질의 효과적인 분석에도 적용 될 수 있을 것이다. 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 (C3Hmod) 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.

Optimal conditions for cytochrome P450 induction in Plutella xylostella (Lepidoptera: Plutellidae)

Ji Hyeong Baek,Si Hyeock Lee 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.10

Cytochrome P450s (P450s) are known to oxidize a variety of insecticides including pyrethroids, thereby conferring metabolic resistance in diamondback moth (DBM), Plutella xylostella. Synergism assay with piperonyl butoxide indicated that the enhanced activity of P450 is associated with pyrethroid resistance in a cypermethrin-resistant (CR) strain. However, there were little differences in the basal transcription levels of all the P450s examined between susceptible (Sus) and CR strains, suggesting that constitutive overexpression of P450 is not likely involved in the cypermethrin resistance but induction of P450 by cypermethrin is rather associated with metabolic resistance. To determine the conditions resulting in maximum levels of P450 induction, several factors including the way of adminstration (topical application vs. leaf dipping), exposure dose and exposure duration were examined. In general, leaf dipping method resulted in greater levels of induction in a wider array of P450s. The conditions of ‘low dose (sublethal dose or concentration) and short exposure (less than 3 hr)' to cypermethrin were more efficient in P450 induction than those of ‘high dose (around LD50 or LC50) and long exposure (more than overnight)’, which have been employed in many other studies to date. Cross-strain comparison revealed that 9 of 11 P450s were induced 1.4-2.2 fold in CR whereas only 3 P450s in Sus under the optimal induction conditions, demonstrating that metabolic resistance in CR strain is actually conferred by the mechanism of selective P450 induction when exposed to cypermethrin.