Molecular Consequences and Rescue of Defect

This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM081568]; the Robert A. Welch Foundation [Endowed Chair AQ-0012] (to B. S. S. M.); and the Fundacao para a Ciencia e a Tecnologia (Portugal) [Grant PTDC/SAU-GMG/71911/2006].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 similar to 2 mu M. As determined by FMN fluorescence quenching, Y181D had K-d(FMN) = 7.3 mu M. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of similar to 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 mu M 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 mu M 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.publishersversionpublishe