SciCombinator

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Concept: Cytochrome P450 reductase

171

Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.

Concepts: P450-containing systems, Catalysis, Cytochrome P450 reductase, Electrochemistry, Nitrogen, Enzyme, Metabolism, Cytochrome P450

166

We have previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 system, through the hepatic deletion of either cytochrome P450 oxidoreductase (POR; HRN line) or cytochrome b5 (Cyb5; HBN line). However, HRN mice still exhibit low levels of mono-oxygenase activity, in spite of the absence of detectable reductase protein. To investigate whether this is because cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the P450 system, we have crossed HRN with HBN mice to generate a line lacking hepatic expression of both electron donors (HBRN). HBRN mice exhibited exacerbation of the phenotypic characteristics of the HRN line - liver enlargement, hepatosteatosis and increased expression of certain cytochrome P450s. Also, drug metabolising activities in vitro were further reduced relative to the HRN model, in some cases to undetectable levels. Pharmacokinetic studies in vivo demonstrated that midazolam half-life, Cmax and area under the concentration-time curve (AUC) were increased, and clearance was decreased, to a greater extent in the HBRN line than in either the HBN or HRN model. Microsomal incubations using NADPH concentrations below the apparent Km of cytochrome b5 reductase, but well above that for POR, led to the virtual elimination of 7-benzyloxyquinoline turnover in HRN samples. These data provide strong evidence that cytochrome b5/cytochrome b5 reductase can act as a sole electron donors to the cytochrome P450 system in vitro and in vivo.

Concepts: Paracetamol, CYP3A4, P450-containing systems, Cytochrome P450 reductase, Cytochrome P450

28

Our aim was to characterize Adverse Drug Reactions (ADRs) related to drug-drug interactions (DDIs) related to involvement of cytochrome P450 (CYP450) isoenzymes in a pharmacovigilance database.

Concepts: Cytochrome P450 reductase, Pharmacology, Cytochrome P450, Adverse drug reaction

28

NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase (NOS), two members of the diflavin oxidoreductase family, are multi-domain enzymes containing distinct FAD and FMN domains connected by a flexible hinge. FAD accepts a hydride ion from NADPH, and reduced FAD donates electrons to FMN, which in turn transfers electrons to the heme center of cytochrome P450 or NOS oxygenase domain. Structural analysis of CYPOR, the prototype of this enzyme family, has revealed the exact nature of the domain arrangement and the role of residues involved in cofactor binding. Recent structural and biophysical studies of CYPOR have shown that the two flavin domains undergo large domain movements during catalysis. NOS isoforms contain additional regulatory elements within the reductase domain that control electron transfer through Ca(2+)-dependent calmodulin (CaM) binding. The recent crystal structure of an iNOS Ca(2+)/CaM-FMN construct, containing the FMN domain in complex with Ca(2+)/CaM, provided structural information on the linkage between the reductase and oxgenase domains of NOS, making it possible to model the holo iNOS structure. This review summarizes recent advances in our understanding of the dynamics of domain movements during CYPOR catalysis and the role of the NOS diflavin reductase domain in the regulation of NOS isozyme activities.

Concepts: Nitric oxide, Metabolism, Nitric oxide synthase, Cytochrome P450, Enzymes, Cytochrome P450 reductase, Electron, Enzyme

27

Interindividual variability in cytochrome P450 (CYP)-mediated xenobiotic metabolism is extensive. CYP metabolism requires two electrons, which can be donated by NADPH cytochrome P450 oxidoreductase (CYPOR) and/or cytochrome b5 (b5). Although substantial number of studies have reported on the function and effect of b5 in CYP-mediated catalysis, its mode of action is still not fully understood.

Concepts: Xenobiotic metabolism, Enzyme, CYP1A2, Xenobiotic, P450-containing systems, Cytochrome P450 reductase, Metabolism, Cytochrome P450

23

Cytochrome P450s are associated with metabolizing of a wide range of compounds including insecticides. CYP353D1v2 has been found over-expressed in the imidacloprid-resistant strain of Laodelphax striatellus. Thus, this study was conducted to express CYP353D1v2 in Sf9 cell as a recombinant protein, to assess its ability in metabolizing imidacloprid.

Concepts: Steroid, Escherichia coli, Protein, Cytochrome P450 reductase, Endoplasmic reticulum, DNA, Metabolism, Cytochrome P450

3

Paraquat, a herbicide linked to Parkinson’s disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology.

Concepts: Gene, Bacteria, Hydrogen peroxide, P450-containing systems, Mitochondrion, Cytochrome P450 reductase, Oxidative phosphorylation, Cytochrome P450

0

Inconsistent conclusions have been reported for the genetic relationship between CYP4F2 (Cytochrome P450 Family 4 Subfamily F Member 2) polymorphisms and the susceptibility to cardiovascular and cerebrovascular diseases.

Concepts: Cytochrome P450 reductase, CYP4F22, Cytochrome P450

0

Cytochrome P450s (P450s) comprise one of the largest known protein families. They occur in every kingdom of life and catalyze essential reactions, such as carbon source assimilation, synthesis of hormones and secondary metabolites, or degradation of xenobiotics. Due to their outstanding ability of specifically hydroxylating complex hydrocarbons, there is a great demand to use these enzymes for biocatalysis, including applications at an industrial scale. Thus, the recombinant production of these enzymes is intensively investigated. However, especially eukaryotic P450s are difficult to produce. Challenges are faced due to complex cofactor requirement as well as the availability of a redox-partner (cytochrome P450 reductase, CPR) are key elements to get active P450s. Additionally, most eukaryotic P450s are membrane bound which complicates the recombinant production. This review describes current strategies for expression of P450s in the microbial cell factories Escherichia coli , Saccharomyces cerevisiae and Pichia pastoris.

Concepts: Cytochrome P450 reductase, Protein, Brewing, Escherichia coli, Bacteria, Fungus, Cytochrome P450, Metabolism

0

Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.

Concepts: Enzyme, P450-containing systems, Glycogen, In vitro, Liver, Cytochrome P450 reductase, Metabolism, Cytochrome P450