Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase

Christopher C. Marohnic, Satya P. Panda, Pavel Martásek, Bettie Sue Masters

Research output: Contribution to journalArticle

42 Citations (Scopus)

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 ∼1:1:1, whereas ∼1:0.1:0.9 was observed for V492E, which retained 9% of the WT k cat/Km 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 ω-hydroxylation of prostaglandin E 1 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.

Original languageEnglish (US)
Pages (from-to)35975-35982
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number47
DOIs
StatePublished - Nov 24 2006

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Antley-Bixler Syndrome Phenotype
NADPH-Ferrihemoprotein Reductase
Flavin-Adenine Dinucleotide
Cytochrome P-450 Enzyme System
Oxidoreductases
Hydroxylation
Assays
Flavin Mononucleotide
Heme Oxygenase (Decyclizing)
Disorders of Sex Development
Mutation
Gene encoding
Xenobiotics
Prostaglandins E
Anchors
Polymorphism
Oxidation-Reduction
Purification
Catalytic Domain
Protein Isoforms

ASJC Scopus subject areas

  • Biochemistry

Cite this

Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase. / Marohnic, Christopher C.; Panda, Satya P.; Martásek, Pavel; Masters, Bettie Sue.

In: Journal of Biological Chemistry, Vol. 281, No. 47, 24.11.2006, p. 35975-35982.

Research output: Contribution to journalArticle

Marohnic, Christopher C. ; Panda, Satya P. ; Martásek, Pavel ; Masters, Bettie Sue. / Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 47. pp. 35975-35982.
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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 ∼1:1:1, whereas ∼1:0.1:0.9 was observed for V492E, which retained 9{\%} of the WT k cat/Km 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 ω-hydroxylation of prostaglandin E 1 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.",
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T1 - Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase

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AU - Panda, Satya P.

AU - Martásek, Pavel

AU - Masters, Bettie Sue

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AB - 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 ∼1:1:1, whereas ∼1:0.1:0.9 was observed for V492E, which retained 9% of the WT k cat/Km 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 ω-hydroxylation of prostaglandin E 1 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.

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