Modulation of Human CYP19A1 Activity by Mutant NADPH P450 Oxidoreductase

Abstract

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.

Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

“…This finding is corroborated by their wild-type-like cytochrome c reduction efficiency (Table 1), except for variant P228L, which demonstrated 45% loss of this activity. Variants classified in this second group have been identified in individuals with altered steroidogenesis (catalyzed by CYP17A1, CY19A1, and CYP21A1) (Huang et al, 2005;Dhir et al, 2007;Pandey et al, 2007). Our results with these variants indicate that these mutations may produce P450-dependent effects, explainable by the location of the P228L mutation (interaction domain with protein redox partners), but to a lesser extent for the M263V, A287P, and G413S mutations (hinge region/connecting domain).…”

Genetics of P450 oxidoreductase: Sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations

“…The figure was created with Molsoft ICM. capacity of our initially described mutants to support the activity of aromatase (P450aro) (30). Thus, multiple studies show that the activity of a specific POR missense mutant as assayed with one P450 cannot be extrapolated to infer its activity with other P450s.…”

“…The structure of rat POR, determined crystallographically, indicates that the electrondonating FMN domain interacts with the redox-partner binding site of a P450 by electrostatic interactions (24). Modeling of these interactions with cytochrome c (24) and with P450 enzymes (30) suggests that the POR residues that interact with the electron recipient may vary among various P450s. This variation in POR/ P450 contact sites would suggest that POR missense mutants that alter POR conformation might impair activity to differing degrees, depending on the electron recipient used to assay activity.…”

Theoretical Study of the Mechanism of Exemestane Hydroxylation Catalyzed by Human Aromatase Enzyme

“…[12][13][14][15][16] In this catalytic process, each oxidation step consumes one mol of NADPH, one mol of molecular oxygen and requires of the presence of the cytochrome P450 reductase (CPR) as a source of electrons. 10,[17][18][19][20][21][22] The overall process of aromatization of androgens via the enzyme aromatase has been depicted in Scheme 1.…”