title: Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase.
abstract: Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia. CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of approximately 1:1:1, whereas approximately 1:0.1:0.9 was observed for V492E, which retained 9% of the WT k(cat)/K(m) in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed omega-hydroxylation of prostaglandin E1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS.
authors.affiliation: Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Marohnic
authors.forename: Satya P
authors.initials: SP
authors.lastname: Panda
authors.forename: Pavel
authors.initials: P
authors.lastname: Martásek
authors.forename: Bettie Sue
authors.initials: BS
authors.lastname: Masters
mesh_terms: D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003398:Craniosynostoses / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000502:physiology; D010084:Oxidation-Reduction; D011485:Protein Binding; D013577:Syndrome
chemical_list: D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D009251:NADPH-Ferrihemoprotein Reductase