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Structure-Functional Analysis of Human Cytochrome P450 2C8 Using Directed Evolution.

ABSTRACT: The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug-drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were ~310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6α-hydroxylation showed that the mutants exhibited a 5-7-fold increase in k_cat values and a 3-5-fold increase in catalytic efficiencies (k_cat/K_M). In arachidonic acid epoxidation, two mutants exhibited a 30-150-fold increase in k_cat values and a 40-110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower K_d value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.

SUBMITTER: Lee R

PROVIDER: S-EPMC8469462 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cytochrome P450 2C8 pharmacogenetics: a review of clinical studies.

Project description:Cytochrome P450 (CYP) 2C8 is responsible for the oxidative metabolism of many clinically available drugs from a diverse number of drug classes (e.g., thiazolidinediones, meglitinides, NSAIDs, antimalarials and chemotherapeutic taxanes). The CYP2C8 enzyme is encoded by the CYP2C8 gene, and several common nonsynonymous polymorphisms (e.g., CYP2C8*2 and CYP2C8*3) exist in this gene. The CYP2C8*2 and *3 alleles have been associated in vitro with decreased metabolism of paclitaxel and arachidonic acid. Recently, the influence of CYP2C8 polymorphisms on substrate disposition in humans has been investigated in a number of clinical pharmacogenetic studies. Contrary to in vitro data, clinical data suggest that the CYP2C8*3 allele is associated with increased metabolism of the CYP2C8 substrates, rosiglitazone, pioglitazone and repaglinide. However, the CYP2C8*3 allele has not been associated with paclitaxel pharmacokinetics in most clinical studies. Furthermore, clinical data regarding the impact of the CYP2C8*3 allele on the disposition of NSAIDs are conflicting and no definitive conclusions can be made at this time. The purpose of this review is to highlight these clinical studies that have investigated the association between CYP2C8 polymorphisms and CYP2C8 substrate pharmacokinetics and/or pharmacodynamics in humans. In this review, CYP2C8 clinical pharmacogenetic data are provided by drug class, followed by a discussion of the future of CYP2C8 clinical pharmacogenetic research.

| S-EPMC2778050 | biostudies-literature

Structure of Human Cytochrome P450 2C19, a Critical Enzyme for Drug Metabolism♦ : Structural Characterization of Human Cytochrome P450 2C19. Active Site Differences between P450s 2C8, 2C9, and 2C19

Project description: Not available

| S-EPMC3531773 | biostudies-literature

Structural characterization of human cytochrome P450 2C19: active site differences between P450s 2C8, 2C9, and 2C19.

Project description:To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme.

| S-EPMC3531772 | biostudies-literature

Corrections of Frequencies of Cytochrome P450 2B6 and 2C8 Allelic Variants in the Mozambican Population.

Project description: Not available

| S-EPMC5101976 | biostudies-literature

Role of cytochrome P450 2C8 and 2J2 genotypes in calcineurin inhibitor-induced chronic kidney disease.

Project description:ObjectivesThe calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus (Tac) help prevent allograft rejection but are associated with nephrotoxicity. Cytochrome P450 2C8 (CYP2C8) and CYP2J2 are polymorphic enzymes expressed in the kidney that metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids, promoting kidney homeostasis. This study examined the association between CNI-induced nephrotoxicity in liver transplant patients and CYP2C8 and CYP2J2 polymorphisms.MethodsLiver transplantation patients receiving CNIs for at least 3 years were genotyped for CYP2C8*3, CYP2C8*4, CYP2C8 Haplotypes B and C, and CYP2J2*7 and evaluated for nephrotoxicity (serum creatinine > or = 1.6 mg/dl) 3-year post-transplantation. CYP2C8 proteins were also engineered in E. coli and their activity towards AA and inhibition by CNIs was investigated in vitro.ResultsThe risk of kidney disease post-transplantation was positively associated with CYP2C8*3 genotype. Odds ratios for all participants carrying at least one CYP2C8*3 allele were significant [odds ratio=2.38 (1.19-4.78)]. Stratification by CNI indicated a significant association between CYP2C8*3 and nephrotoxicity among patients receiving Tac but not CsA. The risk of renal dysfunction was not significantly influenced by CYP2C8*4, CYP2J2*7, or CYP2C8 haplotype B genotypes although inheritance of haplotype C seems to be protective. In vitro, the gene products of CYP2C8*3 and CYP2C8*4 were deficient in AA epoxidation, retaining 26 and 18% of wild-type activity, respectively. Circulating plasma concentrations of CsA and Tac inhibited CYP2C8 wild-type in vitro epoxidation of AA by 17 and 35%, respectively.ConclusionInheritance of CYP2C8*3 is associated with a higher risk of developing renal toxicity in patients treated chronically with CNIs, and especially Tac, possibly by reducing formation of kidney protecting vasodilatory epoxyeicosatrienoic acids.

| S-EPMC2709795 | biostudies-literature

Cytochrome P450 enzymes

Project description:Physiological plasticity in a polluted system: A case of an uncultured bacterium and its cypome

| PRJEB38781 | ENA

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

Project description:Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17?-hydroxyprogesterone (17?-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17?-OH-progesterone). We found that the 17?-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (? 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.

| S-EPMC5491764 | biostudies-literature

Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes.

Project description:The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 ?4-5 (CYP2A6 and CYP2E1) or this region and ?2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5 Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

| S-EPMC5743060 | biostudies-literature

Cytochrome P450 2C8 ?3-long-chain polyunsaturated fatty acid metabolites increase mouse retinal pathologic neovascularization--brief report.

Project description:Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ?3-long-chain polyunsaturated fatty acids (?3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown.The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ?3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ?3LCPUFA and ?6LCPUFA and antiangiogenic role of sEH in ?3LCPUFA metabolism were corroborated in aortic ring assays.Our results suggest that CYP2C ?3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.

| S-EPMC4005334 | biostudies-literature

Targeting of splice variants of human cytochrome P450 2C8 (CYP2C8) to mitochondria and their role in arachidonic acid metabolism and respiratory dysfunction.

Project description:In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.

| S-EPMC4207977 | biostudies-literature

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