De-orphanization of Cytochrome P450 2R1

Abstract

Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. In the 1920s, McCollum, Mellanby, and Pappenheimer (see Ref. 1) showed that deficiency of vitamin D caused rickets in experimental animals. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2, 3). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4), the most potent hormone was 1␣,25-dihydroxyvitamin D (5, 6), which was synthesized from 25-hydroxyvitamin D in the kidney (7). The molecular mechanism of vitamin D action was manifest with the identification (8) and cDNA cloning (9) of the vitamin D receptor.Hydroxylation reactions catalyzed by cytochrome P450s (CYP) 1 activate and inactivate vitamin D as a ligand for the receptor. 25-Hydroxylation is performed in the liver by two different enzymes, one located in the mitochondria (10), identified as the CYP27A1 sterol 27-hydroxylase (11-15), and a second in microsomes (16, 17), which has not been identified in most species. A second mitochondrial P450 (CYP27B1), for which an encoding cDNA was isolated by expression cloning (18) In the current study, a cDNA library made from hepatic mRNA of mice deficient in the gene encoding the mitochondrial CYP27A1 enzyme was screened using a vitamin D receptorbased, ligand activation assay (18). A single cDNA specifying a microsomal P450 enzyme termed CYP2R1 with previously unknown substrate specificity was identified. The biochemical properties and tissue distribution of CYP2R1 are consistent with this enzyme being the microsomal vitamin D 25-hydroxylase. EXPERIMENTAL PROCEDURESExpression Plasmids-A mouse adrenodoxin cDNA (mAdx, nucleotides 41-772 of GenBank TM /EBI Data Bank accession no. L29123) was amplified by the polymerase chain reaction (PCR) from random hexamer-primed mouse hepatic cDNAs using the following oligonucleotide

Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

“…To this end, the abilities of the normal and mutant enzymes to activate the VDR were evaluated in three reporter gene systems in which VDREs were linked to transcription of a LUC gene. These experiments took advantage of the fact that 25-hydroxyvitamin D 3 is a more potent ligand for the VDR than is nonhydroxylated vitamin D 3 (2). As shown by the data in Fig.…”

“…The insert was then excised by digestion with the restriction enzymes SalI and NotI, and subcloned into an untreated pCMV6 expression vector. by TLC were performed as described (2). A solvent system of cyclohexane:ethyl acetate (3:2, vol͞vol) was used for thin layer chromatography.…”

“…Receptor activation assays were performed as described (2). Briefly, HEK 293 cells were grown in 96-well plates and were transfected with the indicated plasmid DNAs for a period of 8-10 h. Vitamin D ligands were then added to the medium in ethanol, and the cells cultured for an additional [16][17][18][19][20] h. Experiments were terminated by cell lysis and measurements of ␤-galactosidase and luciferase (LUC) enzyme activities with appropriate substrates were performed (2).…”

“…At least six CYPs can catalyze this reaction in vitro, including CYP2C11, CYP2D25, CYP3A4, CYP2J1, CYP27A1, and CYP2R1 (2)(3)(4). Of these CYPs, the two most viable candidates for the vitamin D 25-hydroxylase are CYP27A1 and CYP2R1 (2).…”

Case report: vitamin D‐dependent rickets type 1 caused by a novel CYP27B1 mutation

“…It is an inactive prohormone that can be ingested or synthesized in the skin. The production of the active hormone, calcitriol or 1 α ,25‐dihydroxyvitamin D 3 (1,25(OH) 2 D), results from sequential hydroxylation by members of the cytochrome P‐450 enzymes 1. The regulated step is 1‐hydroxylation (Fig.…”