Human P450 Oxidoreductase Research Articles

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays.

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, we examined approximately 450 missense variants of the POR gene listed in the Genome Aggregation Database (gnomAD) using eleven different in silico prediction tools. We found that 64 novel variants were consistently predicted to be disease-causing by most tools. To validate our findings, we conducted a population analysis and selected two variations in POR for further investigation. The human POR wild type and the R268W and L577P variants were expressed in bacteria and subjected to enzyme kinetic assays using a model substrate. We also examined the activities of several cytochrome P450 proteins in the presence of POR (WT or variants) by combining P450 and reductase proteins in liposomes. We observed a decrease in enzymatic activities (ranging from 35% to 85%) of key drug-metabolizing enzymes, supported by POR variants R288W and L577P compared to WT-POR. These results validate our approach of curating a vast amount of data from genome projects and provide an updated and reliable reference for diagnosing POR deficiency.

Stereoselective Bupropion Hydroxylation by Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase Genetic Variants.

Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by CYP2B6. The metabolite hydroxybupropion derived from t-butylhydroxylation is considered to contribute to the antidepressant and smoking-cessation effects of the parent drug. Bupropion hydroxylation is the canonical in vitro and in vivo probe for CYP2B6 activity. P450 also requires obligate partnership with P450 oxidoreductase (POR). Human CYP2B6 and POR genes are highly polymorphic. Some CYP2B6 variants affect bupropion disposition. This investigation evaluated the influence of several human CYP2B6 and POR genetic variants on stereoselective bupropion metabolism, using an insect cell coexpression system containing CYP2B6, POR, and cytochrome b 5 Based on intrinsic clearances (Clints), relative activities for S,S-hydroxybupropion formation were in the order CYP2B6.4 > CYP2B6.1 > CYP2B6.17 > CYP2B6.5 > CYP2B6.6 ≈ CYP2B6.26 ≈ CYP2B6.19 > CYP2B6.7 > CYP2B6.9 > > CYP2B6.16 and CYP2B6.18; relative activities for R,R-hydroxybupropion formation were in the order CYP2B6.17 > CYP2B6.4 > CYP2B6.1 > CYP2B6.5 ≈ CYP2B6.19 ≈ CYP2B6.26 > CYP2B6.6 > CYP2B6.7 ≈ CYP2B6.9 > > CYP2B6.16 and CYP2B6.18. Bupropion hydroxylation was not influenced by POR variants. CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Though Vmax and Km varied widely among CYP2B6 variants, stereoselectivity was preserved, reflected by similar Clint(S,S-hydroxybupropion)/Clint(R,R-hydroxybupropion) ratios (1.8-2.9), except CYP2B6.17, which was less enantioselective. Established concordance between human bupropion hydroxylation in vitro and in vivo, together with these new results, suggests additional CYP2B6 variants may influence human bupropion disposition. SIGNIFICANCE STATEMENT: Bupropion pharmacokinetics, metabolism, and clinical effects are affected by the CYP2B6*6 polymorphism. Other expressed CYP2B6 polymorphisms had diminished (*5, *6, *7, *9, *19, *26) or defective (*16, *18) in vitro bupropion hydroxylation. P450 oxidoreductase genetic variants had no effect on metabolism, suggesting no clinical consequence of this polymorphism. These CYP2B6 polymorphisms may portend diminished in vivo bupropion hydroxylation and predict additional clinically important variant alleles.

Changes in Steroids, Xenobiotics, and Drug‐Metabolism in Humans by Genetic Variations in NADPH Cytochrome P450 Oxidoreductase

A broad spectrum of human diseases, including disordered steroid metabolism, are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) [1, 2]. Human POR is a diflavin reductase that transfers electrons from NADPH to small molecules, non‐P450 redox partners, and cytochrome P450 proteins in the endoplasmic reticulum [2]. Cytochrome P450 proteins perform an extensive range of reactions, including the metabolism of steroids, drugs, and other xenobiotics. Therefore, genetic variations in POR change several metabolic pathways by changing the activities cytochromes P450 [2]. Due to this unique role of POR in metabolism, it was believed for a long time that genetic defects in POR are unlikely, and indeed a POR knockout mouse is embryonically lethal. However, in 2004, the first human patients with defects in POR were reported, and since then, over 200 different variations in POR have been found [3, 4].Recently, we have characterized POR variations from non‐clinical samples [5]. By analyzing the POR sequences from the 1000 genome and other sequencing projects, we identified potentially disease‐causing variations and characterized these by functional studies using recombinant proteins produced in bacteria, yeast, and mammalian cells [6, 7].Identification of severe effects of POR mutations on both the drug and the steroid metabolizing cytochromes P450 indicates that likely pathogenic mutations may be found in non‐clinical population. Their combination as compound heterozygotes or homozygous may severely impact both steroid and drug metabolism by modifications of cytochrome P450 activities. Variations in POR need to be evaluated individually. Changes in drug and steroid metabolism due to genetic variations can be addressed using personalized metabolic profiling and supplementation using modified dosages of drugs and steroids.Support or Funding InformationSupported by grants from the Swiss National Science Foundation (31003A‐134926) & Novartis Foundation for Medical‐Biological Research (18A053) to AVP.

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.

Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography-mass spectrometry. Michaelis-Menten kinetic parameters were determined. For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Cytochrome P450 oxidoreductase genetic polymorphism and pantoprazole pharmacokinetics in healthy volunteers

ABSTRAKTWstęp: Genetycznie uwarunkowana aktywność enzymów metabolizujących leki może mieć znaczenie dla bezpieczeństwa i skuteczności leczenia. We wcześniejszych badaniach wykazano, że na aktywność enzymatyczną CYP2C19 może wpływać nie tylko polimorfizm genu kodującego ten cytochrom, lecz także zawartość reduktazy cytochromu P450 (POR) w mikrosomach ludzkich hepatocytów. Ludzki gen POR wykazuje dużą zmienność, a allel POR*28 jest związany ze zwiększoną aktywnością reduktazy, co w konsekwencji może prowadzić do zwiększonej aktywności układu CYP P450, w tym enzymu CYP2C19.Celem badania było określenie związku pomiędzy polimorfizmem genu POR a parametrami farmakokinetycznymi pantoprazolu, substratu CYP2C19 w grupie zdrowych ochotników. W analizie uwzględniono także warianty genu CYP2C19.Materiały i metody: U 30 osób, u których wykonano badanie pod kątem występowania jedno­nukleotydowych polimorfizmów: rs4244285 (681G>A, CYP2C19*2), rs12248560 (−806C>T, CYP2C19*17) oraz rs1057868 (31696C>T, POR*28). Stężenia pantoprazolu w osoczu zmierzono metodą wysokosprawnej chromatografii cieczowej w ciągu 8 godz. od przyjęcia pojedynczej doustnej dawki leku (40 mg).Wyniki: Nie stwierdzono istotnych różnic w stężeniu pantoprazolu pomiędzy osobami o różnych genotypach POR (*1/*1, *1/*28 i *28/*28). Analiza wieloczynnikowa wykazała, że genotyp CYP2C19 istotnie wpływał na wartość wszystkich analizowanych parametrów farmakokinetycznych (p < 0.05), podczas gdy genotyp POR nie był istotnie związany z żadnym z nich.Wnioski: Wyniki badania wskazują, że polimorfizm genu POR nie wpływa istotnie na farmakokinetykę pantoprazolu.

Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase.

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Specific regulation of thermosensitive lipid droplet fusion by a nuclear hormone receptor pathway

Nuclear receptors play important roles in regulating fat metabolism and energy production in humans. The regulatory functions and endogenous ligands of many nuclear receptors are still unidentified, however. Here, we report that CYP-37A1 (ortholog of human cytochrome P450 CYP4V2), EMB-8 (ortholog of human P450 oxidoreductase POR), and DAF-12 (homolog of human nuclear receptors VDR/LXR) constitute a hormone synthesis and nuclear receptor pathway in Caenorhabditis elegans This pathway specifically regulates the thermosensitive fusion of fat-storing lipid droplets. CYP-37A1, together with EMB-8, synthesizes a lipophilic hormone not identical to Δ7-dafachronic acid, which represses the fusion-promoting function of DAF-12. CYP-37A1 also negatively regulates thermotolerance and lifespan at high temperature in a DAF-12-dependent manner. Human CYP4V2 can substitute for CYP-37A1 in C. elegans This finding suggests the existence of a conserved CYP4V2-POR-nuclear receptor pathway that functions in converting multilocular lipid droplets to unilocular ones in human cells; misregulation of this pathway may lead to pathogenic fat storage.

Altered CYP19A1 and CYP3A4 Activities Due to Mutations in the Flavin Mononucleotide Binding Domain of Human P450 Oxidoreductase

Altered CYP19A1 and CYP3A4 Activities Due to Mutations in the Flavin Mononucleotide Binding Domain of Human P450 Oxidoreductase

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

Impact on CYP19A1 activity by mutations in NADPH cytochrome P450 oxidoreductase

Cytochrome P450 aromatase (CYP19A1), in human placenta metabolizes androgens to estrogens and uses reduced nicotinamide adenine dinucleotide phosphate through cytochrome P450 oxidoreductase (POR) for the energy requirements of its metabolic activities. Mutations in the human POR lead to congenital adrenal hyperplasia due to loss of activities of several steroid metabolizing enzymatic reactions conducted by the cytochrome P450 proteins located in the endoplasmic reticulum. Effect of POR mutations on different P450 activities depend on individual partner proteins. In this report we have studied the impact of mutations found in the POR on the enzymatic activity of CYP19A1. We expressed wild type as well mutant human POR proteins in bacteria and purified the recombinant proteins, which were then used in an in vitro reconstitution system in combination with CYP19A1 and lipids for enzymatic analysis. We found that several mutations as well as polymorphisms in human POR can cause reduction of CYP19A1 activity. This would affect metabolism of estrogens in people with variations of POR allele. The POR mutants Y181D and R616X were found to have no activity in supporting CYP19A1 reactions. The POR mutations Y607C and delF646 showed a loss of 60–90% activity and two polymorphic forms of POR, R316W and G413S showed similar to WT activity. One POR variant, Q153R had almost double the activity of WT. Loss of CYP19A1 activity may contribute to disordered steroidogenesis in female patients with POR mutations as well as in mothers with POR variants carrying a male child.

Contribution of CYP3A isoforms to dealkylation of PDE5 inhibitors: a comparison between sildenafil N-demethylation and tadalafil demethylenation.

The aim of this study was to characterize the kinetics of metabolite formation of the phosphodiesterase type-5 (PDE5) inhibitors sildenafil and tadalafil by CYP3A4, CYP3A5, and CYP3A7 isoforms. The formations of N-desmethyl sildenafil and desmethylene tadalafil were examined using CYP3A supersomes co-expressing human P450 oxidoreductase and cytochrome b5. Both sildenafil N-demethylation and tadalafil demethylenation were catalyzed by CYP3A4, CYP3A5, and to a lesser extent by CYP3A7. The kinetics of desalkyl metabolite formation of the two drugs were well fitted to the Hill equation; however, the Hill coefficients (n) suggested CYP3A-mediated negative cooperativity. Next, we analyzed the kinetics with a two binding sites model assuming two reaction steps: reaction 1 with high-affinity and low-capacity metabolism and reaction 2 with low-affinity and high-capacity metabolism. The kinetics of desalkyl metabolite formation were also fitted to the two binding sites model. The intrinsic clearance (CLint) values of reactions 1 and 2 for sildenafil N-demethylation were 0.733 and 0.033 µL/min/pmol P450 for CYP3A4, 0.788 and 0.019 µL/min/pmol P450 for CYP3A5, and 0.079 and 0.004 µL/min/pmol P450 for CYP3A7, respectively. The CLint values of reactions 1 and 2 for tadalafil demethylenation were 0.187 and 0.014 µL/min/pmol P450 for CYP3A4, 0.050 and <0.001 µL/min/pmol P450 for CYP3A5, and 0.004 and <0.001 µL/min/pmol P450 for CYP3A7, respectively. These results may provide the basis not only for understanding the metabolic properties of the two PDE5 inhibitors, but also for one possible explanation of the mechanisms of CYP3A-mediated negative cooperativity.

FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells

One-electron reductases that reduce nitro compounds in hypoxic human tumour cells are poorly characterized, but are important for targeting hypoxia with nitroaromatic prodrugs. Fluorogenic probes with defined reductase profiles are needed to interrogate the activity of these enzymes in intact cells. In the present paper, we report a 6-nitroquinolone ester (FSL-61) as a fluorogenic probe for POR (NADPH:cytochrome P450 oxidoreductase) activity under hypoxia, and demonstrate its suitability of monitoring POR by flow cytometry. Reduction of FSL-61 by purified recombinant human POR generated the corresponding hydroxylamine, which was non-fluorescent, but was reduced further to the fluorescent amine in cells. Hydrolysis of the ester side chain facilitated cellular entrapment, enabling detection of heterogeneous POR expression in mixed populations of cells. In addition to POR, forced expression of three other diflavin reductases [MTRR (methionine synthase reductase), NDOR1 (NADPH-dependent diflavin oxidoreductase 1) and NOS2A (nitric oxide synthase 2A)] or NADPH:adrenoredoxin oxidoreductase in HCT116 cells significantly increased hypoxic activation of FSL-61. This reductase profile is similar to that for the dinitrobenzamide prodrug PR-104A under hypoxia, and fluorogenic metabolism of FSL-61 correlated significantly with PR-104A activation in a panel of 22 human tumour cell lines. The present study thus demonstrates the utility of FSL-61 for rapid and non-destructive interrogation of the activity of one-electron reductases in hypoxic cells at the single-cell level.

Pharmacogenetics of P450 oxidoreductase

The redox reaction of cytochrome P450 enzymes (CYP) is an important physiological and biochemical reaction in the human body, as it is involved in the oxidative metabolism of both endogenous and exogenous substrates. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the hepatic microsomal CYP enzymes. It plays a crucial role in drug metabolism and treatment by not only acting as an electron donor involved in drug metabolism mediated by CYP enzymes but also by directly inducing the transformation of some antitumor precursors. Studies have found that the gene encoding human POR is highly polymorphic, which is of considerable clinical significance as it affects the metabolism and curative effects of clinically used drugs. This review aims to discuss the effect of POR and its genetic polymorphisms on drug metabolism and therapy, as well as the potential mechanisms of POR pharmacogenetics.

NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

P450 Oxidoreductase Deficiency: A Disorder of Steroidogenesis with Multiple Clinical ManifestationsA Presentation from the European Society for Paediatric Endocrinology (ESPE) New Inroads to Child Health (NICHe) Conference on Stress Response and Child Health in Heraklion, Crete, Greece, 18 to 20 May 2012.

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[(2)H]-pregnenolone; 17-[(2)H]-, 16α-[(2)H]-, 21,21,21-[(2)H(3)]-, and 21-[(2)H]-progesterone; and 21,21,21-[(2)H(3)]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[(2)H]- and 17-[(2)H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[(2)H(3)]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with (D)V values of 1.4-5.1 and (D)V/K values of 1.8-5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[(2)H]-progesterone with 33-44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly nonlinear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.

Effect of P450 oxidoreductase variants on the metabolism of model substrates mediated by CYP2C9.1, CYP2C9.2, and CYP2C9.3

CYP2C9 is a microsomal cytochrome P450 that receives electrons from P450 oxidoreductase (POR) to metabolize about 15% of clinically used drugs. Similar to many P450 enzymes, CYP2C9 is polymorphic, with the hypomorphic *2 and *3 variants accounting for about 20% of White alleles. POR is also polymorphic, with the amino acid sequence variant A503V accounting for 19-37% of alleles in different populations. We aimed to understand how polymorphisms in these two interacting proteins might affect drug metabolism. We assayed the activities of CYP2C9.1, CYP2C9.2, and CYP2C9.3 to metabolize diclofenac, flurbiprofen, and tolbutamide using a wild type or one of four POR variants (Q153R, A287P, R457H, and A503V). Human CYP2C9 and POR variants were expressed in bacteria, purified, and reconstituted in vitro and the Michaelis constant and maximum velocity were measured with each CYP2C9/POR combination and each substrate. With wild-type POR, the CYP2C9 activities were CYP2C9.1>CYP2C9.2>>CYP2C9.3 with all three substrates. Both the common A503V polymorphism and the rare Q153R variant showed modest increases in activity with all three CYP2C9 isoforms and all three substrates. This is in contrast to previous studies in which A503V showed a modest loss of function with CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP17A1. The disease-causing POR variants A287P and R457H had a very low or unmeasurable activity with all CYP2C9 isoforms and all substrates, which is consistent with their low activities with other CYPs. POR variants affect CYP2C9 activities. The impact of a POR variant on catalysis varies with the isoform of CYP2C9 and the assay substrate.

High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins.

Transcriptional Regulation of the Human P450 Oxidoreductase Gene: Hormonal Regulation and Influence of Promoter Polymorphisms

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon. Common human POR polymorphisms at -208 and -173 had little influence on transcription, but the polymorphism at -152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between -249 and -261 and binding of thyroid hormone receptor-β (TRβ) at -240/-245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRβ, and estrogen receptor-α were bound between -374 and -149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T(3) exerts major tropic effects via TRβ, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T(3) also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRβ and Smad3/4 in its expression and indicating that the common polymorphism at -152 may play a role in genetic variation in steroid biosynthesis and drug metabolism.