title: Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect.
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 approximately 2 microM. As determined by FMN fluorescence quenching, Y181D had K(d)(FMN) = 7.3 microM. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of approximately 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 microM activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_POR(Y181D) membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_POR(WT) membranes with a 1.2 microM 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.
authors.affiliation: Department of Biochemistry, 7760, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. marohnic@uthscsa.edu
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Marohnic
authors.forename: Satya P
authors.initials: SP
authors.lastname: Panda
authors.forename: Karen
authors.initials: K
authors.lastname: McCammon
authors.forename: José
authors.initials: J
authors.lastname: Rueff
authors.forename: Bettie Sue Siler
authors.initials: BS
authors.lastname: Masters
authors.forename: Michel
authors.initials: M
authors.lastname: Kranendonk
mesh_terms: D019943:Amino Acid Substitution; D002273:Carcinogens / Q000378:metabolism; D002942:Circular Dichroism; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D002273:Carcinogens; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C014227:ferricyanide reductase