Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome <i>p</i>-450

Madhok, Thelma C.; DeLuca, Hector F.

doi:10.1042/bj1840491

Cited by 112 publications

(38 citation statements)

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“…5B and 8B), the mitochondrial CYP27A1 enzyme was more active at higher concentrations of vitamin D 3 substrate than was CYP2R1. This behavior represents a third line of evidence in support of CYP2R1 being the microsomal vitamin D 25-hydroxylase because in vivo data (42) and in vitro data (10,17,44) suggest that the mitochondrial hydroxylase is a low affinity, high capacity enzyme, whereas the microsomal hydroxylase is a high affinity, low capacity enzyme. Although the reasons for these apparently different kinetic properties were not determined here, future experiments with the recombinant CYP27A1 and CYP2R1 enzymes should reveal whether they are related to transport of the vitamin into the mitochondrion or microsome, differences in the biochemical characteristics of the two enzymes, or other parameters.…”

Section: Resultsmentioning

confidence: 98%

“…Studies over the last 30 years identified vitamin D 25-hydroxylase activities in both microsomal (16,17) and mitochondrial membranes (10) that were associated with cytochrome P450 enzymes. Purification of the mitochondrial protein (12,13), and subsequent cDNA cloning (14,15,37), yielded the CYP27A1 sterol 27-hydroxylase, which, in addition to hydroxylating vitamin D, catalyzed 27-hydroxylation of sterol intermediates in the bile acid biosynthetic pathway (38).…”

Section: Resultsmentioning

confidence: 99%

“…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)(12)(13)(14)(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), catalyzes 1␣-hydroxylation of 25-hydroxyvitamin D in the kidney, and a third mitochondrial P450, vitamin D 24-hydroxylase (CYP24A1), inactivates the vitamin in this tissue (19 -21).…”

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confidence: 99%

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De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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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

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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De-orphanization of Cytochrome P450 2R1

Cheng

Motola

Mangelsdorf

et al. 2003

Journal of Biological Chemistry

353

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“…Early studies indicated that 25-hydroxylase activity is present in both mitochondrial and microsomal subcellular fractions (25)(26)(27), and that the two enzymes exhibit different biochemical properties with respect to substrate affinity and capacity (28,29). The mitochondrial enzyme was purified (30,31), and after the cloning of its cDNA (32), revealed to be the CYP27A1 CYP that is also responsible for the 27-hydroxylation of sterol intermediates in bile acid synthesis (33).…”

Section: Discussionmentioning

confidence: 99%

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

Cheng

Levine

Bell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

659

399

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The synthesis of bioactive vitamin D requires hydroxylation at the 1␣ and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1␣-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.T he metabolic pathway leading to the synthesis of active vitamin D involves three reactions that occur in different tissues (1). The pathway is initiated in the skin with the UV light-mediated cleavage of 5,7,-cholestadien-3␤-ol to produce the secosteroid (3␤,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol (vitamin D 3 ). The second step occurs in the liver and is catalyzed by a cytochrome P450 (CYP) enzyme that hydroxylates carbon 25, producing the intermediate 25-hydroxyvitamin D 3 , which is the major circulatory form of the vitamin. The third and final step takes place in the kidney and involves 1␣-hydroxylation by another CYP, producing 1␣,25-dihydroxyvitamin D 3 . This product is a potent ligand of the vitamin D receptor (VDR) and mediates most of the physiological actions of the vitamin (1).Although the chemical and enzymatic steps in the vitamin D 3 biosynthetic pathway have been known for 30 years (1), the enzyme catalyzing the 25-hydroxylation step in the liver has never been identified. At least six CYPs can catalyze this reaction in vitro, including CYP2C11, CYP2D25, CYP3A4, CYP2J1, CYP27A1, and CYP2R1 (2-4). Of these CYPs, the two most viable candidates for the vitamin D 25-hydroxylase are CYP27A1 and CYP2R1 (2). Both enzymes are expressed in the liver and are conserved among species known to have an active vitamin D signaling pathway. However, mutations in the human and mouse genes encoding the mitochondrial CYP27A1 protein impair bile acid synthesis, but have no consequences for vitamin D metabolism (5-9). It is thus not clear whether the two vitamin D 3 25-hydroxylases represent an example of biological redundancy in an important biosynthetic pathway or whether CYP2R1 alone or some unidentified enzyme fulfills this essential role.In contrast to the uncertainty surrounding vitamin D 3 25-hydroxylase, the renal enzyme responsible for 1␣-hydroxylation of the vitamin...

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“…It has been established that 25-hydroxylation is catalyzed by cytochrome P450 enzymes present in microsomes and mitochondria of liver (15,16). Until now, one mitochondrial and three microsomal cytochrome P450 enzymes have been reported as vitamin D 3 25-hydroxylases: CYP2C11 (17, 18), CYP2D25 (23,24), and CYP2R1 (25).…”

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confidence: 99%

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Yamasaki

Izumi

Ide

et al. 2004

Journal of Biological Chemistry

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Vitamin D 3 requires the 25-hydroxylation in the liver and the subsequent 1␣-hydroxylation in the kidney to exert its biological activity. Vitamin D 3 25-hydroxylation is hence an essential modification step for vitamin D 3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D 3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D 3 relative to other substrates. In this study, we purified vitamin D 3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D 3 and 1␣-hydroxyvitamin D 3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50 -60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D 3 and 1␣-hydroxyvitamin D 3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D 3 25-hydroxylation in rat liver. The most important physiological function described for 1,25-(OH) 2 D 3 is maintenance of calcium homeostasis (a process that involves calcium absorption from the intestine, reabsorption from the kidney, and mobilization from the bone) (2, 3, 5). In addition, involvement in a variety of processes such as cell differentiation and proliferation has been reported (1, 3). In the 1990s, two important kidney mitochondrial cytochrome P450s involved in vitamin D 3 metabolism were identified. One is CYP27B1, which catalyzes the 1␣-hydroxylation reaction, essential for vitamin D 3 activation. Its expression is regulated by parathyroid hormone, 1,25-(OH) 2 D 3 , and calcitonin (6 -11). The genetic defect of the gene results in pseudovitamin D deficiency rickets (12). The other is CYP24, which directs the catabolism of 1,25-(OH) 2 D 3 and 25-OH-D 3 by the hydroxylation at position C-24. The expression of this gene is primarily controlled by 1,25-(OH) 2 D 3 via a vitamin D receptor (13, 14). These two enzymes are apparently regulated in a reciprocal manner depending on physiological factors concerning calcium homeostasis, by which the biological activity of vitamin D 3 is maintained at appropriate levels in the body (1).In contrast to the 1␣-hydroxylase and 24-hydroxylase in kidney, the 25-hydroxylase of vitamin D 3 in liver remains not fully understood, although 25-OH-D 3 is the major circulation form of vitamin D ...

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Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450

Cited by 112 publications

References 29 publications

De-orphanization of Cytochrome P450 2R1

De-orphanization of Cytochrome P450 2R1

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

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

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