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Inter and intra ethnic variation of vitamin K epoxide reductase complex and cytochrome P450 4F2 genetic polymorphisms and their prevalence in South Indian population.

ABSTRACT: BACKGROUND:Genetic variation in the vitamin K epoxide reductase complex (VKORC1) and cytochrome P450 4F2 (CYP4F2) genes were found to be strongly associated with the oral anticoagulant (OA) dose requirement. The distribution of genetic variation in these two genes was found to show large inter- and intra-ethnic difference. MATERIALS AND METHODS:A total of 470 unrelated, healthy volunteers of South Indians of either sex (age: 18-60 years) were enrolled for the study. A 5 ml of venous blood was collected and the genomic deoxyribonucleic acid (DNA) was extracted by using phenol-chloroform extraction method. Real-time quantitative polymerase chain reaction (RT-PCR) method was used for genotyping. RESULTS:The variant allele frequencies of VKORC1 rs2359612 (T), rs8050894 (C), rs9934438 (T) and rs9923231 (A) were found to be 11.0%, 11.8%, 11.7% and 12.0%, respectively. The variant allele VKORC1 rs7294 was (80.1%) more frequent and the variant allele CYP4F2 * 3 was found to be 41.8% in South Indians. The allele, genotype and haplotype frequencies of VKORC1 and CYP4F2 gene were distinct from other compared HapMap populations (P < 0.0001). CONCLUSION:The findings of our study provide the basic genetic information for further pharmacogenetic based investigation of OA therapy in the population.

SUBMITTER: Kumar DK

PROVIDER: S-EPMC3841554 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Common variants of cytochrome P450 4F2 exhibit altered vitamin E-{omega}-hydroxylase specific activity.

Project description:Human cytochrome P450 4F2 (CYP4F2) catalyzes the ?-hydroxylation of the side chain of tocopherols (TOH) and tocotrienols (T3), the first step in their catabolism to polar metabolites excreted in urine. CYP4F2, in conjunction with ?-TOH transfer protein, results in the conserved phenotype of selective retention of ?-TOH. The purpose of this work was to determine the functional consequences of 2 common genetic variants in the human CYP4F2 gene on vitamin E-?-hydroxylase specific activity using the 6 major dietary TOH and T3 as substrate. CYP4F2-mediated ?-hydroxylase specific activity was measured in microsomal preparations from insect cells that express wild-type or polymorphic variants of the human CYP4F2 protein. The W12G variant exhibited a greater enzyme specific activity (pmol product · min(-1) · pmol CYP4F2(-1)) compared with wild-type enzyme for both TOH and T3, 230-275% of wild-type toward ?, ?, and ?-TOH and 350% of wild-type toward ?, ?, and ?-T3. In contrast, the V433M variant had lower enzyme specific activity toward TOH (42-66% of wild type) but was without a significant effect on the metabolism of T3. Because CYP4F2 is the only enzyme currently shown to metabolize vitamin E in humans, the observed substrate-dependent alterations in enzyme activity associated with these genetic variants may result in alterations in vitamin E status in individuals carrying these mutations and constitute a source of variability in vitamin E status.

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Computational Insight Into Vitamin K<sub>1</sub> ω-Hydroxylation by Cytochrome P450 4F2.

Project description:Vitamin K1 (VK1) plays an important role in the modulation of bleeding disorders. It has been reported that ω-hydroxylation on the VK1 aliphatic chain is catalyzed by cytochrome P450 4F2 (CYP4F2), an enzyme responsible for the metabolism of eicosanoids. However, the mechanism of VK1 ω-hydroxylation by CYP4F2 has not been disclosed. In this study, we employed a combination of quantum mechanism (QM) calculations, homology modeling, molecular docking, molecular dynamics (MD) simulations, and combined quantum mechanism/molecular mechanism (QM/MM) calculations to investigate the metabolism profile of VK1 ω-hydroxylation. QM calculations based on the truncated VK1 model show that the energy barrier for ω-hydroxylation is about 6-25 kJ/mol higher than those at other potential sites of metabolism. However, results from the MD simulations indicate that hydroxylation at the ω-site is more favorable than at the other potential sites, which is in accordance with the experimental observation. The evaluation of MD simulations was further endorsed by the QM/MM calculation results. Our studies thus suggest that the active site residues of CYP4F2 play a determinant role in the ω-hydroxylation. Our results provide structural insights into the mechanism of VK1 ω-hydroxylation by CYP4F2 at the atomistic level and are helpful not only for characterizing the CYP4F2 functions but also for looking into the ω-hydroxylation mediated by other CYP4 enzymes.

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Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

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Project description: Not available

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Endothelial deletion of the cytochrome P450 reductase leads to cardiac remodelling

Project description:The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR-/-). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR-/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR-/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.

2022-12-21 | GSE214776 | GEO

Association of a functional cytochrome P450 4F2 haplotype with urinary 20-HETE and hypertension.

Project description:Cytochrome P450 4F2 (CYP4F2) catalyzes the omega-hydroxylation of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a natriuretic and vasoactive eicosanoid that participates in the development of hypertension. The relationship among CYP4F2 genetic variants in the regulatory region, formation of renal 20-HETE, and hypertension is unknown. Here are reported seven genetic variants around the CYP4F2 intronic regulatory region. Four of these variants made up two common haplotypes, Hap I (c.-91T/c.-48G/c.-13T/c.+34T) and Hap II (c.-91C/c.-48C/c.-13C/c.+34G). Hap I included a major functional variant, c.-91T-->C, which was identified by reporter assay and electrophoretic mobility shift assay. Transfected into HEK293 cells, the Hap I construct showed a trend toward higher basal transcriptional activity and exhibited significantly greater LPS-stimulated activity than Hap II; these findings were the result of different NF-kappaB binding affinity between the two constructs. In vivo, a case-control study demonstrated that homozygosity for Hap I doubled the risk for hypertension in a Chinese population, even after adjustment for risk factors including age, gender, and body mass index. This association was confirmed in a family-based association study. In addition, Hap I was associated with elevated urinary 20-HETE. These results indicate that a functional variant of the CYP4F2 regulatory region, which increases the binding affinity of NF-kappaB, increases the risk for hypertension, likely by modulating the production of 20-HETE.

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Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions.

Project description:Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.

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A regulatory loop involving the cytochrome P450-soluble epoxide hydrolase axis and TGF-b signaling

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The FMN "140s Loop" of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450.

Project description:Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".

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Project description:Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the  membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism.

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