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J. Biol. Chem., Vol. 281, Issue 47, 35975-35982, November 24, 2006

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Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase^*

Christopher C. Marohnic¹, Satya P. Panda¹, Pavel Martásek, and Bettie Sue Masters²

From the Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229 and The Department of Pediatrics, Charles University School of Medicine I, Prague, Czech Republic, 2-121-09

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/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 -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.

Received for publication, July 26, 2006

^* This investigation was supported by National Institutes of Health Grant HL30050 (to B. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, references, Table S1, and Figs. S1-S3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: The Robert A. Welch Distinguished Professor in Chemistry, Dept. of Biochemistry, 7760, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. Tel.: 210-567-6627; Fax: 210-567-6984; E-mail: masters{at}uthscsa.edu.

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