Wild-type P450 Oxidoreductase Research Articles

Isoform‐Dependent Effects of Cytochrome P450 Oxidoreductase Polymorphisms on Drug Metabolism by Cytochrome P450 Enzymes in Dogs

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 (CYP) enzymes. We have recently discovered three polymorphisms in the dog POR coding region resulting in one (POR‐H2 and POR‐H4) or two (POR‐H3) amino acid substitutions. In humans, POR mutations can have CYP isoform‐dependent effects on CYP enzyme activity depending on the POR amino acid residue that is substituted.1 Genotyping of 2,040 DNA samples collected from 65 different dog breeds revealed a heterogeneous distribution of POR alleles across breeds. The POR‐H2 allele was found in 19 of 65 breeds and was most common in Rottweilers and Doberman Pinschers (51% and 19% allele frequencies, respectively). The POR‐H3 allele was found in 8 of 65 breeds and was most common in Scottish Deerhounds and Greyhounds (36% and 35% allele frequencies, respectively). The POR‐H4 allele was rare, only being found in 5 of 2,040 dogs. Greyhounds have been shown to metabolize substrates of CYP2B11 (but not other CYPs including CYP2D15) much more slowly compared with mixed‐breed dogs.2 Preliminary studies identified polymorphisms in the CYP2B11 gene that only partially explained reduced CYP2B11 metabolism in Greyhounds. Consequently, we hypothesized that the newly discovered canine POR variants found in Greyhounds would reduce CYP metabolism in a CYP isoform‐dependent manner. The aim of this study was to determine the functional impact of the canine POR variants on drug metabolism by CYP2B11 and CYP2D15 in vitro. In silico molecular structure modelling predicted that POR‐H3 and POR‐H4 but not POR‐H2 would have altered function. The three POR variants and wild‐type were expressed alone or co‐expressed with CYP2B11 or CYP2D15 using an insect cell system. Enzyme kinetic experiments were conducted to determine the impact of the mutations on POR function and CYP‐catalyzed reactions. The POR variants were not significantly different from wild‐type POR in protein expression or function (cytochrome c reduction) when expressed alone. POR variants also did not significantly alter CYP2D15 protein level or catalytic activities or CYP2B11 protein level when co‐expressed. However, POR‐H3 and POR‐H4 (but not POR‐H2) significantly reduced CYP2B11 catalytic efficiency (CLint) by 37% and 69%, respectively, compared to wild‐type POR. This was due to reduced maximal velocities (Vmax) but not substrate affinities (Km). POR‐H3 and POR‐H4 appear to reduce CYP activities in a CYP isoform‐dependent manner. Further investigations are needed to determine POR variant effects on other CYP isoforms and redox partners. The POR‐H3 and POR‐H4 variants are likely to contribute to slower CYP2B11 drug metabolism in Greyhounds and possibly related sighthound breeds (such as Scottish Deerhounds), but results must be confirmed in vivo.Support or Funding InformationAmerican Kennel Club Canine Health Foundation (Grants 2242 and 2529) and the William R. Jones Endowment at Washington State University College of Veterinary Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype–phenotype and structure–function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Effects of genetic variants of human P450 oxidoreductase on catalysis by CYP2D6 in vitro

Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6. N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (K(m)) and maximum velocity (V(max)) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as V(max)/K(m). Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol. POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40-50%, potentially explaining some genetic variation in drug metabolism.