title: Effect of P450 oxidoreductase variants on the metabolism of model substrates mediated by CYP2C9.1, CYP2C9.2, and CYP2C9.3.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  POR variants affect CYP2C9 activities. The impact of a POR variant on catalysis varies with the isoform of CYP2C9 and the assay substrate.
authors.affiliation: Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Murali
authors.initials: M
authors.lastname: Subramanian
authors.forename: Vishal
authors.initials: V
authors.lastname: Agrawal
authors.forename: Duanpen
authors.initials: D
authors.lastname: Sandee
authors.forename: Harrison K
authors.initials: HK
authors.lastname: Tam
authors.forename: Walter L
authors.initials: WL
authors.lastname: Miller
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Tracy
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000493:pharmacokinetics; D005480:Flurbiprofen / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014044:Tolbutamide / Q000493:pharmacokinetics
chemical_list: D011994:Recombinant Proteins; D004008:Diclofenac; D005480:Flurbiprofen; D014044:Tolbutamide; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase