7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase

Abstract

7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation. These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7 expression indicated that DHCR7 protein levels were elevated 2-fold in POR-null microsomes. Addition of POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based, in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%. Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol reductase activity.

Introduction

7-Dehydrocholesterol reductase (DHCR7) is a 54 kDa microsomal enzyme that catalyzes the final step in cholesterol synthesis, the reduction of the sterol C(7-8) double bond, using NADPH as an electron source. Mutations in the DHCR7 gene that impair or inactivate the enzyme result in Smith-Lemli-Opitz Syndrome (SLOS), a disease characterized by developmental and neurological deficits due to cholesterol deficiency [1]. Human and rat DHCR7 cDNAs were cloned in 1998 and 1999, respectively [2], [3], and the enzymes were expressed in yeast and shown to be active in microsomal preparations. A subsequent study with rat liver microsomes [4] presented several lines of evidence that DHCR7 required NADPH-cytochrome P450 reductase (POR) for activity; this included a marked inhibition of DHCR7 activity in microsomes by addition of antibody to POR, as well as a reconstitution of DHCR7 activity by addition of purified POR to protease-treated microsomes and to partially purified DHCR7 preparations. Although NADPH-cytochrome P450 reductase is more widely recognized for its role in the reduction of cytochrome P450 in the oxidation of xenobiotics and steroids, POR is required for cholesterol synthesis, where it is the requisite electron donor to lanosterol demethylase (CYP51A1) [5] and the principal, but not exclusive, electron donor to squalene monooxygenase [6]. POR is ubiquitously expressed in mammalian cells and also in yeast.

DHCR7 has not yet been purified to homogeneity or expressed in a host lacking POR (e.g., Escherichia coli), so the requirement for POR for activity has not been unequivocally established. Moreover, the absence of a recognizable NADPH binding domain in this protein, or the closely related reductase, Δ14-sterol reductase (TM7SF2) has further clouded the issue. The recent availability of hepatic POR-null mice [7] provided an opportunity to examine the requirement for this electron transfer protein in mammalian microsomes that lack this reductase. Our results argue strongly that POR is not required for DHCR7 activity.