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 language | English (US) |
|---|---|
| Pages (from-to) | 35975-35982 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 281 |
| Issue number | 47 |
| DOIs | |
| State | Published - Nov 24 2006 |
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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 journal › Article
}
TY - JOUR
T1 - Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase
AU - Marohnic, Christopher C.
AU - Panda, Satya P.
AU - Martásek, Pavel
AU - Masters, Bettie Sue
PY - 2006/11/24
Y1 - 2006/11/24
N2 - 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.
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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U2 - 10.1074/jbc.M607095200
DO - 10.1074/jbc.M607095200
M3 - Article
VL - 281
SP - 35975
EP - 35982
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 47
ER -