Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect
- 2 November 2009
- journal article
- Published by American Society for Pharmacology & Experimental Therapeutics (ASPET) in Drug Metabolism and Disposition
- Vol. 38 (2), 332-340
- https://doi.org/10.1124/dmd.109.030445
Abstract
Patients with congenital adrenal hyperplasia, exhibiting combined CYP17 and CYP21 deficiency, were shown by Arlt et al. (2004) to harbor a 541T→G mutation in exon 5 of POR (encoding NADPH-cytochrome P450 reductase, CYPOR), which resulted in a Y181D substitution that obliterated electron transfer capacity. Using bacterial expression models, we examined catalytic and physical properties of the human CYPOR Y181D variant. As purified, Y181D lacked flavin mononucleotide (FMN) and NADPH-cytochrome c reductase (NCR) activity but retained normal flavin adenine dinucleotide binding and NADPH utilization. Titration of the purified protein with FMN restored 64 of wild-type (WT) NCR activity in Y181D with an activation constant of ∼2 μM. As determined by FMN fluorescence quenching, Y181D had KdFMN = 7.3 μM. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of ∼1:10 in the engineered MK_1A2_POR Escherichia coli strain, showed the compromised capacity of Y181D to support CYP1A2-catalyzed metabolism of the procarcinogens 2-aminoanthracene, 2-amino-3-methylimidazo(4,5-f)quinoline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Isolated MK1A2_POR membranes confirmed FMN stimulation of Y181D NCR activity with a 1.6 μM activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_PORY181D membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_PORWT membranes with a 1.2 μM FMN activation constant. Therefore, we conclude that compromised FMN binding is the specific molecular defect causing POR deficiency in patients with Y181D mutation and that this defect, in large part, can be overcome in vitro by FMN addition.Keywords
This publication has 39 references indexed in Scilit:
- Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductaseEMBO Reports, 2009
- Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450Published by Elsevier BV ,2009
- Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay SystemDrug Metabolism and Disposition, 2009
- Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced CytotoxicityDrug Metabolism and Disposition, 2006
- Escherichia coli BTC, a human cytochrome P450 competent tester strain with a high sensitivity towards alkylating agents: involvement of alkyltransferases in the repair of DNA damage induced by aromatic aminesMutagenesis, 2005
- The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformationMutagenesis, 2005
- P450 oxidoreductase deficiency: a new disorder of steroidogenesis with multiple clinical manifestationsTrends in Endocrinology & Metabolism, 2004
- Compound heterozygous mutations of cytochrome P450 oxidoreductase gene (POR) in two patients with Antley–Bixler syndromeAmerican Journal of Medical Genetics Part A, 2004
- Crystal structure of the FMN‐binding domain of human cytochrome P450 reductase at 1.93 Å resolutionProtein Science, 1999
- Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450lmBiochemical and Biophysical Research Communications, 1979