Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreducatse

Pediatric Endocrinology and Diabetology, University of Bern, Freiburgstrasse 15, G3 812, CH-3010 Bern, Switzerland.
Molecular Endocrinology (Impact Factor: 4.02). 11/2007; 21(10):2579-95. DOI: 10.1210/me.2007-0245
Source: PubMed


Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

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Available from: Amit V Pandey, Oct 05, 2015
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    • "The p.C569Y mutation was compound heterozygote together with p.V608F (Flück et al., 2004). The p.V608F retained 57% of 17,20 lyase activity and 80% of 17α-hydroxylase activity (Table 2 and Figure 5) (Huang et al., 2005; Pandey et al., 2007). Both p.C569Y and p.V608F had more severe effects on CYP19A1 activity than on CYP17A1 activities (Flück et al., 2004; Huang et al., 2005; Pandey et al., 2007). "
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    ABSTRACT: Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler syndrome but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations with Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V allele is found in about 30% of all alleles but there are some differences across different population groups.
    Frontiers in Pharmacology 04/2014; 5(103). DOI:10.3389/fphar.2014.00103 · 3.80 Impact Factor
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    • "In vitro functional testing of POR mutant P399_E401del on single enzymes showed an activity loss of 68–85% for different P450s. The severity of aromatase inhibition by P399_E401del (;85%) and in utero virilization may be linked as we have suggested earlier for other mutations in the NADPH-binding domain of POR (Pandey et al., 2007). Computational studies suggest that P399_E401del causes structural instability and may impair electron transfer from NADPH to FAD. "
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    ABSTRACT: This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiological, pharmacological and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene monooxygenase and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell culture model in order to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure-function to the impacts of POR genetic variation on human drug and steroid metabolism.
    Drug metabolism and disposition: the biological fate of chemicals 10/2012; 41(1). DOI:10.1124/dmd.112.048991 · 3.25 Impact Factor
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    • "The hydrophobic N-terminal membrane anchor of ClCPR is essential for its function in the P450 catalytic cycle. It serves to anchor the protein molecule to the endoplasmic reticulum which ensures proper spatial interaction for electron transfer between the ClCPR and cytochrome P450s [41], [46]. Without the hydrophobic anchor, ClCPR is incapable of transferring electrons to cytochrome P450s. "
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    ABSTRACT: NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.
    PLoS ONE 02/2012; 7(2):e31037. DOI:10.1371/journal.pone.0031037 · 3.23 Impact Factor
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