Molecular Cloning and mRNA Expression of Cytochrome P450 (CYP450)-related Protein in the Pacific Oyster, Crassostrea gigas: A Water Temperature and Time Study

Jo, Pil-Gue,Min, Tae-Sun,An, Kwang-Wook,Choi, Cheol-Young The Korean Society for Integrative Biology 2009 Animal cells and systems Vol.13 No.4

We cloned the complete complementary DNA (cDNA) of a Pacific oyster (Crassostrea gigas) cytochrome P450 (CYP450)-related protein using rapid amplification of cDNA ends (RACE). The cDNA included a 1470 bp open reading frame that began with the first ATG codon at position 103 bp and ended with a TAG stop codon at position 1573 bp (GenBank accession EF451959). The sequence had all major functional domains and characteristics of previously characterized CYP450 molecules, including the heme-binding region (FGVGRRRCVG) and putative arginine codon (R) integral to enzymatic function. An NCBI/GenBank database comparison to other CYP450 genes revealed that the deduced C. gigas CYP450 amino acid sequence is similar to that of mouse (Mus musculus) CYP450 2D/II (28%, accession AK078880), rabbit (Oryctolagus cuniculus) CYP450 2D/II (28%, AB008785), and white-tufted-ear marmoset (Callithrix jacchus) CYP450 2D (28%, AY082602). Thus, although the C. gigas CYP450 we cloned appears to belong to the 2D type of the CYP450 group, it has low similarity to this type. CYP450 mRNA expression increased over 6 h in C. gigas gills at $30^{\circ}C$ and $10^{\circ}C$, and then decreased, indicating that CYP450 plays an important role in C. gigas exposed to water temperature changes. This finding can be used as a physiological index for Pacific oysters exposed to changing water temperatures.

Molecular Cloning and mRNA Expression of Cytochrome P450 (CYP450)-related Protein in the Pacific Oyster, Crassostrea gigas: A Water Temperature and Time Study

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

We cloned the complete complementary DNA (cDNA) of a Pacific oyster (Crassostrea gigas) cytochrome P450 (CYP450)-related protein using rapid amplification of cDNA ends (RACE). The cDNA included a 1470 bp open reading frame that began with the first ATG codon at position 103 bp and ended with a TAG stop codon at position 1573 bp (GenBank accession EF451959). The sequence had all major functional domains and characteristics of previously characterized CYP450 molecules, including the heme-binding region (FGVGRRRCVG) and putative arginine codon (R) integral to enzymatic function. An NCBI/GenBank database comparison to other CYP450 genes revealed that the deduced C. gigas CYP450 amino acid sequence is similar to that of mouse (Mus musculus) CYP450 2D9 (29%, accession AK078880), rabbit (Oryctolagus cuniculus) CYP450 2D/II (28%, AB008785), and white-tufted-ear marmoset (Callithrix jacchus) CYP450 2D (28%, AY082602). Thus, although the C. gigas CYP450 we cloned appears to belong to the 2D type of the CYP450 group, it has low similarity to this type. CYP450 mRNA expression increased over 6 h in C. gigas gills at 30ºC and 10ºC, and then decreased, indicating that CYP450 plays an important role in C. gigas exposed to water temperature changes. This finding can be used as a physiological index for Pacific oysters exposed to changing water temperatures. We cloned the complete complementary DNA (cDNA) of a Pacific oyster (Crassostrea gigas) cytochrome P450 (CYP450)-related protein using rapid amplification of cDNA ends (RACE). The cDNA included a 1470 bp open reading frame that began with the first ATG codon at position 103 bp and ended with a TAG stop codon at position 1573 bp (GenBank accession EF451959). The sequence had all major functional domains and characteristics of previously characterized CYP450 molecules, including the heme-binding region (FGVGRRRCVG) and putative arginine codon (R) integral to enzymatic function. An NCBI/GenBank database comparison to other CYP450 genes revealed that the deduced C. gigas CYP450 amino acid sequence is similar to that of mouse (Mus musculus) CYP450 2D9 (29%, accession AK078880), rabbit (Oryctolagus cuniculus) CYP450 2D/II (28%, AB008785), and white-tufted-ear marmoset (Callithrix jacchus) CYP450 2D (28%, AY082602). Thus, although the C. gigas CYP450 we cloned appears to belong to the 2D type of the CYP450 group, it has low similarity to this type. CYP450 mRNA expression increased over 6 h in C. gigas gills at 30ºC and 10ºC, and then decreased, indicating that CYP450 plays an important role in C. gigas exposed to water temperature changes. This finding can be used as a physiological index for Pacific oysters exposed to changing water temperatures.

Comparison of CYP3A4 and CYP3A5: The Effects of Cytochrome b5 and NADPH-cytochrome P450 Reductase on Testosterone Hydroxylation Activities

Lee, S.J.,Goldstein, J.A. 日本藥物動態學會 2012 Drug metabolism and pharmacokinetics Vol.27 No.6

CYP3A4 and CYP3A5 require cytochrome b5 (b5) and NADPH-cytochrome P450 oxidoreductase (CPR) for optimum metabolism, but little is known about the specific requirements for b5 and CPR to produce optimal activities for these enzymes. The metabolism of testosterone (TT) by CYP3A4 and CYP3A5 was analyzed by various combinations of b5 and CPR using a fixed amount of recombinant P450 which had been purified from an Escherichia coli expression system. CYP3A4 and CYP3A5 required 4- and 8-fold more of CPR than of the P450s, respectively, for optimal activity. The requirement of b5 for optimal activity showed the same pattern for both CYP3A4 and CYP3A5, exhibiting a gradual stimulation of the activity reaching a maximum at 16 fold more b5 than P450. Although CYP3A4 exhibited higher activities than CYP3A5 in all combinations, both enzymes exhibited the same dependency profile for b5 and CPR. Therefore, the stronger activity of CYP3A4 compared to CYP3A5 appears to be intrinsic to the CYP3A4 protein itself and not to different requirements for b5 and CPR. Since the relative amounts of b5 and CPR are important in the maintenance of CYP3A4 and CYP3A5 activities, different levels of these proteins in vitro and in vivo may cause altered metabolism of their substrates or misinterpretation of enzyme properties.

인체 간 Microsome에서의 제2세대 Sulfonylurea계 약물들의 Cytochrome P450 약물 대사효소에 대한 억제작용

최지이,김수영,김경아,박지영 대한내분비학회 2002 Endocrinology and metabolism Vol.17 No.4

연구배경: Sulfonylurea계 약물들은 제2형 당뇨병 치료에 널리 사용되는 약물이다. 장기간 투여로 인한 병용투여 약물과의 약물상호작용 가능성이 높고 또한 이들 약물들과 병용투여 약물간의 상호작용으로 인한 독작용이 보고된 예가 있으나 작용기전을 밝힌 연구는 거의 이루어지지 않고 있는 실정이다. 본 연구에서는 약물대사 효소로서 중요한 역할을 하고 있는 cytochrome P450(CYP)에 대해서 제2세대 sulfonylurea계 약물들에 의하여 억제 작용의 가능성이 있는지를 확인하고 이를 통해 약물상호작용의 가능성을 검증하고자 하였다. 방법: 인체간 microxomes를 이용한 glibenclamide, glipizide, 및 gliclazide 등이 CYP1A2, CYP2C9, CYP2C19, CYP2D6 및 CYP3A4 동효소에 대한 억제정도를 각각의 CYP 동효소에 대한 지표약물을 이용하여 농도에 따른 상대적 억제정도로 평가하였다. 결과: Glibenclamide의 경우 CYP2C9에 대한 강한 억제 작용을 보였으며(IC_50, 11.3μM), CYP3A4에 대해서도 중등도의 억제작용이 관찰되었다(IC_50, 5900μM). 또한, CYP1A2, CYP2C19, CYP2D6에 대해서도 억제 작용이 관찰되었으나 상대적으로 미약하였다(IC_50s>112μM). Glipizide는 CYP3A4에 대해서 선택적으로 강한 억제작용을 보였으며(IC_50, 11.2μM) 타 CYP 동 효소에 대해서는 거의 억제 작용을 나타내지 못하였다(IC_50s>500μM). Gliclazide의 경우 CYP2C9에 대해서는 약한 억제작용이 관찰되었으며(IC_50, 276.1μM) 타 CYP 동효소에 대한 억제 작용은 관찰되지 아니하였다(IC_50, >500μM). 결론: CYP 동효소에 대해서 제2세대 sulfonylurea계 약물들이 억제 작용을 나타낼 수 있음을 확인하였으며 이러한 결과는 임상적으로 sulfonylurea와 병용 투여 약물간의 약물 상호작용의 가능성을 제시하고 있다. 그러므로, Sulfonylurea계 약물과 타 약물의 병용 투여시 치료 영역이 좁은 약물이나 농도에 따른 독성이 큰 약물의 경우 적극적인 혈당 조절과 더불어 병용 투여 약물에 대한 적극적인 감시(monitoring)가 필요할 것으로 사료된다. Background: Sulfonylurea drugs have been used for many decades as one of the main families of drugs for the treatment of type 2 diabetes mellitus. Even though there are many opportunities to medicate sulfonylurea drugs concomitantly with many other drugs, and furthermore there have been several case reports on drug interaction with sulfonylurea drugs, there has been no clear demonstration revealing the mechanisms that cause these interactions. We therefore evaluated inhibitory potential of sulfonylurea drugs, including glibenclamide, glipizide and gliclazide, on the cytochrome P450(CYP)-catalyzing enzymes using human liver microsomes. Methods: The inhibitory effects of glibenclamide, glipizide and gliclazide, on the CYP-catalyzing reaction, were evaluated for CYP1A2, CYP2C19, CYP2D6 and CYP3A4 using human liver microsomes, and probe drugs for each. Results: Glibenclamide showed relative potent inhibitory effects on the CYP2C9- and CYP3A4- catallyzed reaction (IC_50; 11.3(μM and 59.0 (μM). The other CYP isoforms tested showed only weak inhibitory effects by due to glibenclamide (IC_50>112(μM). Glipizide showed potent inhibitory effect on CYP3A4-catalyzed reaction only (IC_50; 11.2 (μM), and weak, or no, inhibitory effects on each on the other CYP isoforms tested (IC_50>276 (μM). Conclusion: The sulfonylurea drugs showed inhibitory potential on the CYP-catalyzing reaction in human liver microsomes. The results obtained in the present study provide insights into the potential of the drug interaction to ward drugs co-administered with sulfonylureas. It will be necessary to take into consideration the control of blood glucose, as well as therapeutic drug monitoring, to reduced toxicities when sulfonylurea drugs are co-administered with drugs of a narrow therapeutic range, or with severe dose-dependent toxicities (J Kor Soc Endocrinol 17:544∼553, 2002).

Functional Characterization of Allelic Variants of Polymorphic Human Cytochrome P450 2A6 (CYP2A6*5, *7, *8, *18, *19, and *35)

Han, Songhee,Choi, Seunghye,Chun, Young-Jin,Yun, Chul-Ho,Lee, Chang Hoon,Shin, Hee Jung,Na, Han Sung,Chung, Myeon Woo,Kim, Donghak Pharmaceutical Society of Japan 2012 Biological & pharmaceutical bulletin Vol.35 No.3

<P>Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in <I>Escherichia coli</I> and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F—G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased <I>K</I><SUB>m</SUB> values whereas CYP2A6*18 and *19 showed decreased <I>k</I><SUB>cat</SUB> values, which resulted in lower catalytic efficiencies (<I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB>). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased <I>K</I><SUB>m</SUB> value, whereas CYP2A6*18 and *35 showed much greater decreases in <I>k</I><SUB>cat</SUB> values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.</P>

암브록솔과 세티리진의 Cytochrome P450 저해 활성 평가

김봉희,류창선,장힘찬,이상윤,이지윤,채정우,권광일,김상겸 대한약학회 2013 약학회지 Vol.57 No.3

In the present study we evaluated drug-drug interaction potential of ambroxol and cetirizine mediated by inhi-bition of CYP isoforms including CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 using pooled human liver microsomes (HLMs). As measured by liquid chromatography-electrospray ionization tandem mass pec- trometry, cetirizine and ambroxol inhibited significantly CYP2E1 but the maximal inhibition was approximately 36% at 10 µM cetirizine and 28% at 3 µM ambroxol. In addition, CYP2D6 activity was decreased to approximately 83% of ontrol activity in pooled HLM incubated with 3 µM ambroxol. Activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were not significantly inhibited by cetirizine and ambroxol. Considering their maximal plasma concentration in human (Cmax of cetirizine is approximately 0.67 µM and Cmax of ambroxol is 0.044 µM), these two drugs have very low pos- sibility in drug-drug interaction by CYP inhibition in clinical situations.

Structural insights into the binding of lauric acid to CYP107L2 from <i>Streptomyces avermitilis</i>

Han, Songhee,Pham, Tan-Viet,Kim, Joo-Hwan,Lim, Young-Ran,Park, Hyoung-Goo,Jeong, Dabin,Yun, Chul-Ho,Chun, Young-Jin,Kang, Lin-Woo,Kim, Donghak Elsevier 2017 Biochemical and biophysical research communication Vol.482 No.4

<P><B>Abstract</B></P> <P> <I>Streptomyces avermitilis</I> is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from <I>S. avermitilis</I> shares a high sequence similarity with the PikC (CYP107L1) from <I>S. venezuelae</I>. To elucidate the structural features of CYP107L2, we conducted biochemical and structural characterization of CYP107L2 from <I>S. avermitilis</I>. The CYP107L2 gene was cloned, and its recombinant protein was expressed and purified. The CYP107L2 showed a low-spin state of heme, and the reduced form yielded the CO difference spectra with a maximal absorption at 449 nm. Binding of pikromycin and lauric acid yielded the typical type I spectra with <I>K</I> <SUB>d</SUB> values of 4.8 ± 0.3 and 111 ± 9 <I>μ</I>M, respectively. However, no metabolic product was observed in the enzyme reaction. X-ray crystal structures of the ligand-free CYP107L2 and its complex with lauric acid were determined at the resolution of 2.6 and 2.5 Å, respectively. CYP107L2 showed a well-conserved CYP structure with a wide-open substrate-binding cavity. The lauric acid is bound mainly via hydrophobic interactions with the carboxylate group of lauric acid coordinated to the heme of P450. Glu-40 and Leu-382 residues in the CYP107L2 complex with lauric acid showed significant conformational changes to provide plentiful room for the lauric acid in the substrate-binding site.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CYP107L2 from <I>S. avermitilis</I> shares a high sequence similarity with the PikC. </LI> <LI> Purified CYP107L2 yielded a typical type I binding spectra to pikromycin and lauric acid. </LI> <LI> Crystal structure of CYP107L2 complex with lauric acid was determined at 2.5 Å resolution. </LI> <LI> Laurate is bound mainly via hydrophobic interactions in the substrate binding cavity of CYP107L2. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Cloning, expression and characterization of CYP102D1, a self‐sufficient P450 monooxygenase from <i>Streptomyces avermitilis</i>

Choi, Kwon‐,Young,Jung, EunOk,Jung, Da‐,Hye,Pandey, Bishnu Prasad,Yun, Hyungdon,Park, Hyung‐,yun,Kazlauskas, Romas J.,Kim, Byung‐,Gee Blackwell Publishing Ltd 2012 FEBS JOURNAL Vol.279 No.9

<P>Among 33 cytochrome P450s (CYPs) of <I>Streptomyces avermitilis</I>, CYP102D1 encoded by the <I>sav575</I> gene is naturally a unique and self‐sufficient CYP. Since the native cyp102D1 gene could not be expressed well in <I>Escherichia coli</I>, its expression was attempted using codon‐optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a <I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB> value of 453.4 ± 181.5 μ<SMALL>m</SMALL><SUP>−1</SUP>·min<SUP>−1</SUP>. Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a <I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB> value of 100.9 ± 10.4 μ<SMALL>m</SMALL><SUP>−1</SUP>·min<SUP>−1</SUP> (15‐fold).</P>

CYP3A5*3 Polymorphism and Its Clinical Implications and Pharmacokinetic Role

Ji-Young Park 대한임상약리학회 2014 Translational and Clinical Pharmacology Vol.22 No.1

The cytochrome P450 (CYP) 3A subfamily is estimated to participate in the biotransformation of 50% of the currently prescribed drugs. Four members of the CYP3A subfamily have been identified in humans: CYP3A4, CYP3A5, CYP3A7, and CYP3A43. Initial data suggested that CYP3A5 accounts for only a small proportion of the total hepatic CYP3A in about 20% of samples, but it was later revealed that CYP3A5 represents more than 50% of the total CYP3A amount in some individuals. Several genetic variants have been described for the CYP3A5 gene, of which the CYP3A5*3 allele (gA6986G), the most common form and leading to the loss of CYP3A5 activity, has been extensively investigated in the aspect of pharmacokinetics and disease risk. This review summarized the molecular characteristics of the CYP3A5 gene, and discusses the association of the CYP3A5*3 polymorphism with disease risks such as cancer and hypertension, along with its role in the pharmacokinetics of CYP3A substrates.

Cytochrome P450 2C8 and CYP3A4/5 are Involved in Chloroquine Metabolism in Human Liver Microsomes

Kyoung-AhKim,Ji-YoungPark,Ji-SukLee,SabinaLim 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.8

Chloroquine has been used for many decades in the prophylaxis and treatment of malaria. It is metabolized in humans through the N-dealkylation pathway, to desethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ), by cytochrome P450 (CYP). However, until recently, no data are available on the metabolic pathway of chloroquine. Therefore, the metabolic pathway of chloroquine was evaluated using human liver microsomes and cDNA-expressed CYPs. Chloroquine is mainly metabolized to DCQ, and its Eadie-Hofstee plots were biphasic, indicating the involvement of multiple enzymes, with apparent Km and Vmax values of 0.21 mM and 1.02 nmol/min/mg protein 3.43 mM and 10.47 nmol/min/mg protein for high and low affinity components, respectively. Of the cDNA-expressing CYPs examined, CYP1A2, 2C8, 2C19, 2D6 and 3A4/5 exhibited significant DCQ formation. A study using chemical inhibitors showed only quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4/5 inhibitor) inhibited the DCQ formation. In addition, the DCQ formation significantly correlated with the CYP3A4/5-catalyzed midazolam 1-hydroxylation (r=0.868) and CYP2C8-catalyzed paclitaxel 6a-hydroxylation (r = 0.900). In conclusion, the results of the present study demonstrated that CYP2C8 and CYP3A4/5 are the major enzymes responsible for the chloroquine N-deethylation to DCQ in human liver microsomes.