Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

Christopher C Marohnic, Satya P. Panda, Karen M. McCammon, J. Rueff, Bettie Sue Sile Masters, Michel Kranendonk

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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 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.
Original languageUnknown
Pages (from-to)332-340
JournalDrug Metabolism And Disposition
Volume38
Issue number2
DOIs
Publication statusPublished - 1 Jan 2010

Keywords

  • ANTLEY-BIXLER-SYNDROME
  • MUTANT P450 OXIDOREDUCTASE
  • FMN-BINDING DOMAIN
  • ESCHERICHIA-COLI
  • DISORDERED STEROIDOGENESIS
  • P-450 REDUCTASE
  • TESTER STRAIN
  • RAT-LIVER
  • SYSTEM
  • RIBOFLAVIN

Cite this

Marohnic, Christopher C ; Panda, Satya P. ; McCammon, Karen M. ; Rueff, J. ; Masters, Bettie Sue Sile ; Kranendonk, Michel. / Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect. In: Drug Metabolism And Disposition. 2010 ; Vol. 38, No. 2. pp. 332-340.
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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 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.",
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Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect. / Marohnic, Christopher C; Panda, Satya P.; McCammon, Karen M.; Rueff, J.; Masters, Bettie Sue Sile; Kranendonk, Michel.

In: Drug Metabolism And Disposition, Vol. 38, No. 2, 01.01.2010, p. 332-340.

Research output: Contribution to journalArticle

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T1 - Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

AU - Marohnic, Christopher C

AU - Panda, Satya P.

AU - McCammon, Karen M.

AU - Rueff, J.

AU - Masters, Bettie Sue Sile

AU - Kranendonk, Michel

N1 - info:eu-repo/grantAgreement/FCT/3599-PPCDT/71911/PT# 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].

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N2 - 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.

AB - 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.

KW - strain

KW - antley-bixler-syndrome

KW - escherichia-coli

KW - steroidogenesis

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KW - disordered

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KW - p-450

KW - reductase

KW - fmn-binding

KW - system

KW - rat-liver

KW - domain

KW - mutant

KW - oxidoreductase

KW - riboflavin

KW - ANTLEY-BIXLER-SYNDROME

KW - MUTANT P450 OXIDOREDUCTASE

KW - FMN-BINDING DOMAIN

KW - ESCHERICHIA-COLI

KW - DISORDERED STEROIDOGENESIS

KW - P-450 REDUCTASE

KW - TESTER STRAIN

KW - RAT-LIVER

KW - SYSTEM

KW - RIBOFLAVIN

U2 - 10.1124/dmd.109.030445

DO - 10.1124/dmd.109.030445

M3 - Article

VL - 38

SP - 332

EP - 340

JO - Drug Metabolism And Disposition

JF - Drug Metabolism And Disposition

SN - 0090-9556

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