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

Abstract

P450 oxidoreductase (POR) is an electron-donating flavoprotein required for the activity of all microsomal cytochrome P450 enzymes. We sequenced 5,655 bp of the POR gene in a representative population of 842 healthy unrelated individuals in four ethnic groups: 218 African Americans, 260 Caucasian Americans, 179 Chinese Americans, and 185 Mexican Americans. One hundred forty SNPs were detected, of which 43 were found in >1% of alleles. Twelve SNPs were in the POR promoter region. Fifteen of 32 exonic variations altered the POR amino acid sequence; 13 of these 15 are previously undescribed missense variations. We found eight indels, only one of which was in the coding region. A previously described variant, A503V, was found on 27.9% of all alleles with some ethnic predilection (19.1% in African Americans, 26.4% in Caucasian Americans, 36.7% Chinese Americans, and 31.0% in Mexican Americans). We built cDNA expression vectors for the 13 previously undescribed missense variants, expressed each protein lacking 27 N-terminal residues in Escherichia coli, and assayed the apparent K m and Vmax of each in four assays: reduction of cytochrome c, oxidation of NADPH, 17␣-hydroxylase activity of P450c17, and 17,20 lyase activity of P450c17. The catalytic activities of several missense mutants differed substantially in these assays, indicating that each POR mutant must be assayed separately with each potential target P450 enzyme. The activity of A503V was reduced to a modest but statistically significant degree in all four assays, suggesting that it may play an important role in interindividual variation in drug response.cytochrome P450 ͉ drug metabolism ͉ electron transfer ͉ pharmacogenomics ͉ steroidogenesis P 450 oxidoreductase (POR) is a single protein containing both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) moieties that transfers electrons from NADPH to microsomal (type II) cytochrome P450 enzymes (for review, see ref. 1) (Fig. 1). Cytochrome P450 enzymes (http://drnelson.utmem.edu/ CytochromeP450.html) are heme-containing proteins that catalyze a broad range of oxidative reactions. The human genome contains 57 genes for cytochrome P450 enzymes; 7 encode Type I (mitochondrial) P450s and 50 encode Type II P450s, found in the endoplasmic reticulum (2). Among the 50 human microsomal P450 enzymes, approximately 20 are involved in the biosynthesis of steroids, sterols, fatty acids, and eicosanoids, approximately 15 participate in hepatic drug metabolism, and approximately 15 are ''orphan'' P450 enzymes whose catalytic roles are unclear (3).POR transfers electrons to three steroidogenic enzymes: P450c17 (17␣-hydroxylase/17,20 lyase), P450c21 (21-hydroxylase), and P450aro (aromatase) (4). Individuals with POR deficiency have a broad range of steroidogenic disorders, extending from infants with ambiguous genitalia and skeletal malformations to women with the polycystic ovary syndrome (5-8). Finding severe POR mutations in human patients was unexpected, because POR knockout mice die during embryonic devel...

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

“…Our long-standing biochemical research into the mechanism of this critical flavoprotein (12) led us to explore the structure/ function relationships responsible for these phenotypes by expressing and purifying POR variants of interest reported in the literature (13)(14)(15)(16)(17)(18)(19)(20). The variant A287P was reported as the most common mutation found in the Caucasian population (10,14), and these studies were verified and extended to establish genotype-phenotype correlations in a cohort of patients from 11 countries (21).…”

The POR rs1057868–rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients

“…Thus, the contribution of common POR variants to variability of tacrolimus metabolism is of great interest. POR*28, the most common sequence variant in POR gene, induces an amino acid substitution (C>T, p.Ala503Val) and influence the electron binding moiety of POR [23] . A previous in vitro study showed that this mutation could decrease CYP3A4 activity.…”

P450-Based Nano-Bio-Sensors for Personalized Medicine

“…P450 oxidoreductase complex operates as follows ( Figure 11): electrons from reduced NADPH (released as NADP+), are taken up by the FAD moiety, which is reduced to FADH 2 . Then FADH 2 reduces FMN to FMNH 2 , which in turn passes its two electrons to the CYP heme group, which is associated with the P450 oxidoreductase by electrostatic interactions (Huang et al, 2008). This electron transfer becomes a current of electron that P450 oxidoreductase must supply in the presence of high substrate concentrations.…”

“…Adapted from (Miller, W.L., 2005). Reprinted with permission from Huang et al, 2008. Copyright (2008 National Academy of Sciences, U.S.A. Figure 12) are electron transport hemeproteins which, similarly to cytochrome P450, are built around a central heme group.…”

“…11. Relationship of P450 oxidoreductase to a microsomal cytochrome P450 enzyme (Huang et al, 2008). ER is the endoplasmic reticulum and CYTO is the cytoplasm.…”