Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: Molecular consequences and rescue of defect

Christopher C. Marohnic, Satya P. Panda, Karen McCammon, José Rueff, Bettie Sue Siler Masters, Michel Kranendonk

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

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-POR Y181D 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.

Original languageEnglish (US)
Pages (from-to)332-340
Number of pages9
JournalDrug Metabolism and Disposition
Volume38
Issue number2
DOIs
StatePublished - Feb 2010

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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