Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase.

Sameer S Udhane,Amit V Pandey,Norio Kagawa,Shaheena Parween

doi:10.3389/fphar.2017.00580

Abstract

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Highlights

Cytochrome P450 proteins metabolize a wide range of drugs, steroids and xenobiotics (Schuster and Bernhardt, 2007; Omura, 2010; Zanger and Schwab, 2013)
This loss of activity indicates that these mutations affect electron transport in P450 oxidoreductase (POR) through flavins to cytochrome c since reduction of cytochrome c requires the participation of both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) as do the cytochromes P450
The total flavin content of the gain of function mutation Q153R (1 mol FMN/mol of POR and 0.99 mol FAD/mol of POR) was comparable to WT and for the loss of function mutation P284L the FMN content was similar to WT (0.91 mol FMN/mol of POR) while the FAD content was reduced by 25% (0.75 mol FAD/mol of POR) suggesting that these mutations do not severely affect either the FMN or the FAD binding to POR (Figure 4)

Summary

Introduction

Cytochrome P450 proteins metabolize a wide range of drugs, steroids and xenobiotics (Schuster and Bernhardt, 2007; Omura, 2010; Zanger and Schwab, 2013). There are two distinct types of cytochrome P450 proteins in humans (Omura, 2010). Type 1 P450s, that are located in the mitochondria, are primarily involved in the metabolism of steroids and use Altered CYP19A1 by POR Variations adrenodoxin/adrenodoxin reductase proteins as redox partners (Omura, 2006; Zalewski et al, 2016). POR is a flavoprotein that contains both the flavin mononucleotide (FMN) and the flavin adenine dinucleotide (FAD) and supplies electrons to cytochromes P450 and other proteins (Matsubara et al, 1976; Nagao et al, 1983; Guengerich, 2005). The structure of the FMN binding domain of human POR has been determined by x-ray crystallography (Zhao et al, 1999) and a more recent open conformation structure of POR shows potential interaction possibilities for redox partners (Aigrain et al, 2009)

Methods

Results

Conclusion