Porcine Microsomal Vitamin D3 25-Hydroxylase (CYP2D25)

Hosseinpour, Fardin; Wikvall, Kjell

doi:10.1074/jbc.m004185200

Cited by 77 publications

(18 citation statements)

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“…One was the CYP2D25 enzyme purified from pig liver (34); however, this protein is not well conserved among species, as would be expected for an important vitamin D biosynthetic enzyme, and the closest human homologue (CYP2D6) does not have 25-hydroxylase activity (34). Furthermore, a molecular analysis by another group of the human CYP2D6 in the patient studied here failed to find mutations in the gene (35).…”

Section: Discussionmentioning

confidence: 91%

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.

661

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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Section: Discussionmentioning

confidence: 91%

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.

661

399

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“…For example, rat CYP2C11 is a male-specific enzyme with this activity but is unique to this species and does not act on vitamin D 2 (48,49). Pig CYP2D25 25-hydroxylates both vitamin D 2 and D 3 but there is no corresponding orthologue for this P450 in humans (50). The human CYP2D6 protein shares the greatest sequence identity (77%) with the pig CYP2D25, but the CYP2D6 enzyme does not possess vitamin D 25-hydroxylase activity (50).…”

Section: Resultsmentioning

confidence: 99%

“…Pig CYP2D25 25-hydroxylates both vitamin D 2 and D 3 but there is no corresponding orthologue for this P450 in humans (50). The human CYP2D6 protein shares the greatest sequence identity (77%) with the pig CYP2D25, but the CYP2D6 enzyme does not possess vitamin D 25-hydroxylase activity (50). In contrast to these findings, the CYP2R1 enzyme reported here is not sexually dimorphic, is conserved from fish to humans, and acts on both forms of vitamin D. Sequence identity between the human CYP2R1 and these other rat, pig, and human vitamin D 25-hydroxylase enzymes varies between 33 and 39%, as would be expected from their inclusion in the CYP2 family (51).…”

Section: Resultsmentioning

confidence: 99%

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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“…It is known that a reaction that is catalyzed by a P450 enzyme of a certain subfamily in a distinct species is not necessarily performed by the isoenzyme of the same subfamily in another species. In this context, it is interesting to note that CYP2D25 can catalyze the 25-hydroxylation of vitamin D 3 , whereas CYP2D6 is not able to perform this reaction (Hosseinpour and Wikvall, 2000). Commercially available recombinant P450s expressed in lymphoblasts (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4), which are involved in several xenobiotic metabolisms, were not capable of reducing the model compound benzamidoxime (data not shown) as well as the melagatran prodrugs ximelagatran and N-hydroxy-melagatran.…”

Section: Biotransformation Of Ximelagatran An Amidoxime Prodrugmentioning

confidence: 99%

Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester Prodrug

Clement¹,

Lopian²

2003

Drug Metabolism and Disposition

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b 5 , NADH-cytochrome b 5 reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b 5 and b 5 reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.Melagatran is the active form of the novel, oral thrombin inhibitor ximelagatran (Gustafsson et al., 2001). Melagatran has suboptimal bioavailability because of the presence of a carboxylic acid, a secondary amine, and an amidine residue resulting in a charged molecule at physiological pH values. The prodrug ximelagatran was developed with a view to improving absorption. Ximelagatran comprises an ethyl ester group in place of the carboxylic acid and an N-hydroxyamidine group in place of the amidine. After protonation of amidines at the double-bonded nitrogen, cations are formed that are highly stabilized by mesomerism. Amidines are very strong bases (Albert et al., 1948) and protonated under physiological conditions. By introduction of an oxygen atom in the amidine functional group, the basicity is lowered by 5 pK a value. Amidoximes have been used as prodrugs for certain amidine-containing drugs, including pentamidine derivatives (Clement, 1993) and sibrafiban (Weller et al., 1996). It has been shown that certain amidoximes are rapidly reduced in vitro and in vivo to the amidines (Clement et al., 1988(Clement et al., , 1992Hauptmann et al., 1988). More recently, it has been demonstrated that the model compound benzamidoxime is reduced by microsom...

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Porcine Microsomal Vitamin D3 25-Hydroxylase (CYP2D25)

Cited by 77 publications

References 31 publications

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

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

De-orphanization of Cytochrome P450 2R1

Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester Prodrug

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