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25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

ABSTRACT: A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.

SUBMITTER: Dahlback H

PROVIDER: S-EPMC1149125 | biostudies-other | 1988 May

REPOSITORIES: biostudies-other

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Project description:The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.

Project description:Metabolic deactivation of 1,25(OH)2D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. CYP24A1 function relies on electron transfer from the soluble ferredoxin protein adrenodoxin (Adx). Recent structural evidence suggests that regioselectivity of the CYP24A1 reaction may correlate with distinct modes of Adx recognition. Here we used nuclear magnetic resonance (NMR) spectroscopy to monitor the structure of 15N-labeled full-length Adx from rat while forming the complex with rat CYP24A1 in the ligand-free state or bound to either 1,25(OH)2D3 or the vitamin-D supplement 1α(OH)D3. Although both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a crosslinking analysis suggested that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key finding is that, whereas the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site α-helix 3 of rat adrenodoxin, the interaction in the presence of 1α(OH)D3, which is lacking the carbon-25 hydroxyl, results in disruption of the NMR peak broadening pattern, thus indicating a ligand-induced nonspecific protein interaction. These findings provide a structural basis for the poor substrate turnover of side-chain-modified vitamin-D analogs, while also confirming that specificity of the CYP24A1-ligand interaction influences specificity of CYP24A1-Adx recognition. SIGNIFICANCE STATEMENT: Mitochondrial cytochrome P450 enzymes, such as CYP24A1 responsible for catabolizing vitamin-D and its analogs, rely on a protein-protein interaction with a ferredoxin in order to receive delivery of the electrons required for catalysis. In this study, we demonstrate that this protein interaction is influenced by the enzyme-ligand interaction that precedes it. Specifically, vitamin-D missing carbon-25 hydroxylation binds the enzyme active site with high affinity but results in a loss of P450-ferredoxin binding specificity.

Project description:RationalePatients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS).MethodsBalb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined.ResultsMice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A.ConclusionsVD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.

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25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

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