Review
Genetic variation in human P450 oxidoreductase

https://doi.org/10.1016/j.mce.2008.09.017Get rights and content

Abstract

Catalysis by all 50 Type II (microsomal) P450 enzymes, including steroidogenic P450c17, P450c21, and P450aro and hepatic drug-metabolizing enzymes requires electron donation from P450 oxidoreductase (POR). POR knockout mice are embryonic lethal, but human POR mutations cause a complex disorder of steroidogenesis. Disorders of hepatic drug metabolism in human POR deficiency have not yet been described. To understand the potential contribution of POR to pharmacogenetics, we sequenced the POR gene in 842 normal persons from 4 ethnic groups. We detected 140 single nucleotide sequence variants of which 43 were in >1% of alleles, including 15 missense mutants; this brings the total of known POR missense mutants to 35. A503V was found on 28% of alleles, varying from 19% in African Americans to 37% in Chinese Americans. We expressed all 35 missense mutants in E. coli and assayed their activities to: oxidize NADPH, reduce cytochrome c, support the 17α-hydroxylase and 17,20 lyase activities of bacterially expressed human P450c17, and support the metabolism of fluorogenic EOMCC by bacterially expressed human CYP1A2 and CYP2C19. These data show that there are great differences in the activities of some POR mutants depending on the electron recipient assayed; for example, Q153R causes severely impaired steroid biosynthesis in human patients and in vitro, but is a gain-of-function mutant with CYP1A2 and 2C19. A503V reduces both activities of P450c17 in half, but had no effect on CYP1A2 or 2C19. POR variants are a previously unappreciated source of genetic variation in patterns of steroid synthesis and drug metabolism.

Introduction

Cytochrome P450 enzymes catalyze a broad array of oxidative reactions. Type I P450 enzymes, found in bacteria and in mitochondria, receive electrons from NADPH through a flavoprotein (ferredoxin reductase) and an iron/sulfur protein (ferredoxin); Type II enzymes, found in the endoplasmic reticulum, receive electrons from NADPH through a single protein, P450 oxidoreductase (POR) (for review see Miller, 2005). The human genome contains 57 genes encoding P450 enzymes. Seven of these are for Type I enzymes and 50 encode Type II enzymes. Among these 50 Type II P450s, about 15 are involved in drug metabolism, 20 are involved in the biosynthesis of sterols, fatty acids and eicosanoids and 15 are “orphan” enzymes, whose activities remain unknown or ill-defined (Guengerich, 2004). Steroidogenic Type II P450 enzymes include P450c17 (17α-hydroxylase/17,20 lyase), P450c21 (21-hydroxylase) and P450aro (aromatase). Disruption of POR should affect all Type II P450 enzymes, suggesting it would have disastrous consequences, so that POR mutations were not sought in patients. Consistent with this view, disruption of the POR gene in knockout mice results in early embryonic lethality (Shen et al., 2002, Otto et al., 2003). By contrast, liver-specific knockouts of POR result in a profound disruption of hepatic drug metabolism, but these mice are morphologically and reproductively normal, indicating that the lethality is caused by lack of activity of extra-hepatic P450 enzymes (Henderson et al., 2003, Wu et al., 2003, Gu et al., 2003). A hypomorphic POR mouse expressing only 5–25% of normal amounts of POR has limited embryonic lethality and decreased drug metabolism despite an apparent compensatory increase in total liver P450 content (Wu et al., 2005). Thus one would have predicted that severe mutations of human POR would be lethal.

In 1985, a clinical report described a patient with genital ambiguity and an abnormal urinary steroid profile suggesting partial combined deficiencies of what was then thought to be three distinct steroidogenic enzymes: 17α-hydroxylase, 17,20 lyase, and 21-hydroxylase (Peterson et al., 1985). As these steroidogenic enzyme activities were known to be catalyzed by Type II cytochrome P450 enzymes, we suggested that this patient had a disorder in the electron donor for these enzymes, P450 oxidoreductase (Miller, 1986). Although several similar patients were reported subsequently, this hypothesis lay dormant, and the human POR gene was never cloned. However, the description of the human POR gene though the human genome project, plus the advent of rapid, inexpensive PCR-based sequencing permitted us to examine the POR gene in such patients, resulting in the first four reported cases of POR deficiency (Flück et al., 2004). Since that time, about 50 additional patients have been reported (for review see Scott and Miller, 2008).

Although the initial patient described in 1985 did not have skeletal malformations (Peterson et al., 1985), the majority of patients described to date with POR deficiency also had a pattern of skeletal malformations termed Antley–Bixler syndrome (ABS). This disorder is characterized by craniosynostosis (premature fusion of bones of the skull), radio-ulnar or radio-humeral synostosis, bowed femora and other, more variable skeletal disorders (Antley and Bixler, 1975, Crisponi et al., 1997). Several other congenital malformation syndromes, including Apert, Crouzon, Jackson-Weiss, Pfeiffer and Beare-Stevenson syndromes are also characterized by craniosynostosis. Studies between 1994 and 1996 identified dominant, gain-of-function mutations in the gene for fibroblast growth factor receptor 2 (FGFR2) in these syndromes (Reardon et al., 1994, Muenke et al., 1994, Jabs et al., 1994, Wilkie et al., 1995, Przylepa et al., 1996) including identical missense mutations in several of these (Rutland et al., 1995, Meyers et al., 1996, Schaefer et al., 1998), indicating that these syndromes are variants of a single disease. Examination of several individual patients with ABS also identified mutations in the FGFR2 gene (Chun et al., 1998, Tsai et al., 2001), but in the series of multiple ABS patients, only 7 of 16 had FGFR2 mutations (Reardon et al., 2000). Retrospective review shows that these 7 patients had normal genitalia and no evidence of disordered steroidogenesis, but the 9 patients without FGFR2 mutations had genital anomalies and/or abnormal steroid profiles (Reardon et al., 2000). This study concluded that two genes were involved in each ABS patient (digenic inheritance). However, our sequencing of both the FGFR2 and POR genes in a large group of patients demonstrated that ABS was really two distinct genetic disorders: one due to dominant, gain-of-function FGFR2 mutations (and hence a variant of the other craniosynostosis syndromes caused by gain-of-function mutations in FGFR2), and the other due to recessive mutations in POR (Huang et al., 2005). Thus recessive POR mutations cause the form of ABS that includes genital malformations (in both sexes) and disordered steroidogenesis; we have also found milder POR mutations in people with disordered steroidogenesis who do not have ABS, suggesting that disorders of POR might be rather common (Flück et al., 2004, Huang et al., 2005). A recent report found the POR mutation G539R in a family previously reported to have isolated 17,20 lyase deficiency (Hershkovitz et al., 2008).

Section snippets

P450 oxidoreductase

POR is an 82-kDa membrane-bound protein containing 680 residues. The structure of POR is well understood from the 2.6 Å-resolution crystal structure of N-terminally deleted (N-56) rat POR (Wang et al., 1997). POR has two distinct domains; one contains the NADPH-binding site and the FAD-binding domain, the other contains the FMN domain that eventually interacts with the redox-partner binding site of the P450. Thus POR is the only factor required to transport electrons from NADPH to a microsomal

Variation in the human POR gene

The human genome project located the POR gene on chromosome 7q11.2 and provided the sequence of its 15 protein-coding exons; this information has been used to identify POR mutations in patients with ABS. However, the corresponding cDNA sequence in the database included 5′ untranslated sequences not found in “Exon 1,” and it was known that the rat POR gene contained a 56-base untranslated exon 30.5 kb upstream from the first coding exon (O’Leary et al., 1994). Using the sequence of this rat

Influence of POR mutations on drug metabolism

Although the clinical reports of patients with POR deficiency have not mentioned defective drug metabolism, the essential role of POR with drug-metabolizing hepatic P450 enzymes would imply such an effect, and liver-specific POR knockout mice have a profound defect in hepatic drug metabolism (Henderson et al., 2003, Gu et al., 2003, Wu et al., 2003). Thus we sought to examine the activities of POR mutants with hepatic drug-metabolizing P450 enzymes. For our initial studies we employed CYP1A2

Conclusions

POR is essential for the function of all microsomal P450 enzymes, hence studies of POR biochemistry and genetics impinge upon endocrinology, pharmacology, developmental biology and possibly other areas as well. While it is now clear that severe POR mutations cause the rare, recessive form of ABS, the frequency and phenotypes of mild defects in POR remain unclear. Mild POR defects have been associated with infertility without skeletal malformation and hence may be relatively common. It is clear

Acknowledgements

Supported by NIH grant GM 73020 to WLM. We thank all the patients, families and normal subjects who participated in these studies.

References (49)

  • K.A. O’Leary et al.

    NADPH cytochrome P-450 oxidoreductase gene: identification and characterization of the promoter region

    Arch. Biochem. Biophys.

    (1994)
  • A.V. Pandey et al.

    Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

    J. Biol. Chem.

    (2003)
  • A.V. Pandey et al.

    Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

    J. Biol. Chem.

    (2005)
  • A.L. Shen et al.

    Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c

    J. Biol. Chem.

    (1995)
  • A.L. Shen et al.

    Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome P450 oxidoreductase

    J. Biol. Chem.

    (2002)
  • V. Agrawal et al.

    Pharmacogenetics of P450 oxidoreductase: effect of sequence variants on activities of CYP1A2 and CYP2C19

    Pharmacogenet. Genomics

    (2008)
  • R. Antley et al.

    Trapezoidocephaly, midfacial hypoplasia and cartilege abnormalities with multiple synostoses and skeletal fractures

    Birth Defects Orig. Article Ser.

    (1975)
  • R.J. Auchus et al.

    Molecular modeling of human P450c17 (17α-hydroxylase/17,20-lyase): Insights into reaction mechanisms and effects of mutations

    Mol. Endocrinol.

    (1999)
  • K. Chun et al.

    FGFR2 mutation associated with clinical manifestations consistent with Antley–Bixler syndrome

    Am. J. Med. Genet.

    (1998)
  • G. Crisponi et al.

    Antley–Bixler syndrome: case report and review of the literature

    Clin. Dysmorphol.

    (1997)
  • D.R. Davydov et al.

    Association of cytochromes P450 with their reductases: opposite sign of the electrostatic interaction in P450BM-3 as compared with the microsomal 2B4 system

    Biochemistry

    (2000)
  • C.E. Flück et al.

    Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley–Bixler syndrome

    Nat. Genet.

    (2004)
  • D.H. Geller et al.

    The genetic and functional basis of isolated 17,20 lyase deficiency

    Nat. Genet.

    (1997)
  • D.H. Geller et al.

    P450c17 mutations R347H and R358Q selectively disrupt 17,20-lyase activity by disrupting interactions with P450 oxidoreductase and cytochrome b5

    Mol. Endocrinol.

    (1999)
  • Cited by (43)

    • P450 oxidoreductase deficiency

      2023, Genetic Steroid Disorders: Second Edition
    • P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms

      2017, Journal of Steroid Biochemistry and Molecular Biology
      Citation Excerpt :

      When the POR mutations were found in patients with disordered steroid metabolism and then several variations in POR gene were identified in normal healthy individuals, we proposed that variations in POR may cause alterations in the activities of drug metabolizing cytochrome P450s because the POR is required for nearly all cytochrome P450s located in the endoplasmic reticulum [6,8,12,89]. Several groups have now examined the effect of POR variations on activities of drug metabolizing enzymes [26,27,32,90–97] as well as other POR dependent reactions [25–27,94,96,98,99]. Cytochrome P450 3A4 (CYP3A4) is the major hepatic enzyme that metabolizes a large percentage of drugs and endogenous substrates [4,100,101].

    • P450 Oxidoreductase Deficiency (PORD)

      2014, Genetic Steroid Disorders
    • NADPH P450 oxidoreductase: Structure, function, and pathology of diseases

      2013, Pharmacology and Therapeutics
      Citation Excerpt :

      Several POR variants identified from patients and normal population have now been tested for different enzymatic activities (Flück & Miller, 2004; Huang et al., 2005; Flück et al., 2007; Pandey et al., 2007; Flück et al., 2008). While initial studies focused on steroid metabolizing P450s, several recent studies reported effects of POR mutations on drug metabolizing P450s (Agrawal et al., 2008; Hart et al., 2008; Kranendonk et al., 2008; Gomes et al., 2009; Miller et al., 2009; Oneda et al., 2009; Agrawal et al., 2010; Flück et al., 2010; Marohnic et al., 2010; Nicolo et al., 2010; Tomalik-Scharte et al., 2010). The overall physiological role of POR mutations beyond congenital adrenal hyperplasia (e.g. effects on hepatic drug metabolism, heme oxygenase and direct metabolism of small molecules) are slowly becoming clear as more studies are coming out (Agrawal et al., 2010; Flück et al., 2010; Marohnic et al., 2010; Nicolo et al., 2010; Pandey et al., 2010; Sandee et al., 2010; X. Zhang et al., 2011).

    • Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization

      2011, Archives of Biochemistry and Biophysics
      Citation Excerpt :

      Many microsomal enzymes, including more than four dozen type II cytochrome P450 monooxygenases (P450s) [14], squalene monooxygenase [15], cytochrome b5 [16], fatty acid desaturase [17], and 7-dehydrocholesterol reductase [18] and heme oxygenase [19], form productive redox complexes with CYPOR, in which electron transfer is required for catalysis. Loss of CYPOR function, therefore, results in a complex mixture of deficiencies in endobiotic and xenobiotic metabolism and can lead to severe developmental malformations including midface hypoplasia, humeroradial synostosis, bowing and fracture of femora, and sexual dimorphisms [20]. The formation of biliverdin IXα by heme oxygenase-1 requires three moles of oxygen for the three monooxygenation steps and electrons from NADPH supplied by CYPOR [21], thereby emphasizing the impact of CYPOR deficiencies on this system.

    • Steroids in the Laboratory and Clinical Practice

      2023, Steroids in the Laboratory and Clinical Practice
    View all citing articles on Scopus
    View full text