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 ϳ1:1:1, whereas ϳ1:0.1:0.9 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 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. Antley-Bixler syndrome (ABS)3 is a disorder characterized by severe midface hypoplasia, humeroradial synostosis, bowing and fracture of femora, and other severe developmental malformations (1). The disease was initially attributed to fibroblast growth factor receptor 2 (FGFR2) mutations (2).Miller's group discovered a correlation between six allelic variants of the POR gene (encoding CYPOR, EC 1.6.2.4) and disordered steroidogenesis observed in patients with and without ABS (3). The variants were A287P, R457H, V492E, C569Y, V608F, and an intron 6 splice variant resulting in a premature stop codon. The wild-type human CYPOR cDNA and mutants, created by site-directed mutagenesis, were expressed in Escherichia coli. The resultant proteins were assayed for their respective NADPH-cytochrome c and NADPH-cytochrome P450 reductase activities. The authors concluded that deleterious POR mutations correlated well with the decreased lanosterol demethylase (CYP51) activity that had previously been observed in ABS patients, and that severe POR mutations were sufficient to cause the ABS phenotype, even in the absence of FGFR2 mutations. Retardation of somite and limb bud formation, which was observed previously in embryonic lethal CYPOR knock-out mice (4, 5), seems to substantiate this assertion. The authors also hypothesized that POR mutations were more common than the relatively low incidence of ABS suggested and that milder mutations could result in disordered steroidogenesis or incr...

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

“…At this juncture, the only human mutations that have been shown to be reversible at the molecular level, when examined by addressing the purified, homogeneous enzymes with biophysical techniques, have been those exhibiting flavin deficiencies (22,25,26,38). However, it has become apparent from this work that certain missense mutations could lead to conformational alterations resulting in unstable or more readily degradable proteins, thereby leading to diminished activity, but not necessarily directly affecting flavin or NADPH binding.…”

“…Cell harvest and lysis were as described previously (25) except that the detergent concentration was increased from 0.1 to 0.5% Triton X-100, and the extraction volume ratio was 0.2 mg of original pellet weight/1 ml of 0.5% Triton X-100 extraction buffer to facilitate the extraction of the A287P protein. The extracted protein was affinity-purified on a 2Ј,5Ј-ADP-Sepharose 4B (GE Healthcare) column as described previously (40), with the following modifications.…”

“…and Purification Conditions-We have expressed full-length WT human P450 reductase and 11 POR mutants in E. coli and purified them to homogeneity (22,(25)(26)(27). 10 Our initial purification of the full-length A287P variant followed our established protocol, but the yield was extremely low and irreproducible as compared with WT.…”

“…1B). Protein-bound flavin content, both FAD and FMN, was measured using HPLC (25) for both WT and A287P. The ratio of FAD and FMN to protein was calculated to be ϳ1:1:1, suggesting the mutation has, at most, a minimal effect on flavin content (Table 2).…”

Genetics of P450 oxidoreductase: Sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations

“…Because most drugs used in clinical practice are metabolized by hepatic microsomal (Type II) P450 enzymes that require POR (12,13), one would predict that human POR mutants would impair drug metabolism and/or bile acid biosynthesis. Disorders of these systems have not yet been reported in patients, but POR mutants Y459H and V492E, found in patients (5) disrupt binding of FAD and have impaired activity with CYP4A4 (14). Sequence variations in some Type II P450 enzymes can cause variations in drug responses, both among individuals and among ethnic groups (12,13).…”