Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes.
25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH)2D3 and 24,25(OH)2D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (Vmax/Km) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD+ or NADP+. This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a Km for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD+ suggests that the enzyme has a tightly bound NAD+ in its active site that is released only upon its reduction.
Tuckey RC
，Tang EKY
，Maresse SR
，Delaney DS
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Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4.
20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3.
Cheng CY
，Slominski AT
，Tuckey RC
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Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human CYP24A1.
CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)2D3] as the major and most characterized metabolites. Both display immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20(OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human CYP24A1 to metabolize 20(OH)D3 and 20,23(OH)2D3. Both isoforms metabolized 20(OH)D3 to the same dihydroxyvitamin D species with no secondary metabolites being observed. Hydroxylation at C24 produced both enantiomers of 20,24(OH)2D3. For rat CYP24A1 the preferred initial site of hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH)2D3 was initially metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous to the catabolism of 1,25(OH)2D3 via the C24-oxidation pathway. Similar catalytic efficiencies were observed for the metabolism of 20(OH)D3 and 20,23(OH)2D3 by human CYP24A1 and were lower than for the metabolism of 1,25(OH)2D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas 20,23(OH)2D3 undergoes multiple oxidations which include cleavage of the side chain.
Tieu EW
，Li W
，Chen J
，Kim TK
，Ma D
，Slominski AT
，Tuckey RC
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