Article

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1)

School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
The Journal of steroid biochemistry and molecular biology (Impact Factor: 4.05). 02/2010; 119(3-5):171-9. DOI: 10.1016/j.jsbmb.2010.02.022
Source: PubMed

ABSTRACT CYP27B1 catalyzes the 1alpha-hydroxylation of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3, the hormonally active form of vitamin D3. To further characterize mouse CYP27B1, it was expressed in Escherichia coli, purified and its activity measured on substrates incorporated into phospholipid vesicles, which served as a model of the inner mitochondrial membrane. 25-Hydroxyvitamin D3 and 25-hydroxyvitamin D2 in vesicles underwent 1alpha-hydroxylation with similar kinetics, the catalytic rate constants (k(cat)) were 41 and 48mol/min/mol P450, respectively, while K(m) values were 5.9 and 4.6mmol/mol phospholipid, respectively. CYP27B1 showed inhibition when substrate concentrations in the membrane were greater than 4 times K(m), more pronounced with 25-hydroxyvitamin D3 than 25-hydroxyvitamin D2. Higher catalytic efficiency was seen in vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin than for dimyristoyl phosphatidylcholine vesicles. CYP27B1 also catalyzed 1alpha-hydroxylation of vesicle-associated 24R,25-dihydroxyvitamin D3 and 20-hydroxyvitamin D3, and 25-hydroxylation of 1alpha-hydroxyvitamin D3 and 1alpha-hydroxyvitamin D2, but with much lower efficiency than for 25(OH)D3. This study shows that CYP27B1 can hydroxylate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 associated with phospholipid membranes with the highest activity yet reported for the enzyme. The expressed enzyme has low activity at higher concentrations of 25-hydroxyvitamin D in membranes, revealing that substrate inhibition may contribute to the regulation of the activity of this enzyme.

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    • "and others found no significant difference between the calciferol formulations in their effect in lowering parathyroid hormone (PTH) [2] [7]. Since 1,25-dihydroxyvitamin D (1,25(OH) 2 D) also regulates PTH, the discordant effects of cholecalciferol and ergocalciferol therapy on total 25OHD and PTH could be explained by differences in the catalytic rate of 1-α-hydroxylation by CYP27B1 of the respective metabolites, or differences in substrate inhibition [8]. Most circulating 25OHD and 1,25(OH) 2 D is bound with high affinity to vitamin D-binding protein (DBP), and only a small fraction remains unbound [9]. "
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    • "The rate of CYP27B1 activity in mitochondria of living cells is unknown but CYP27B1 enzymatic activity has been measured in reconstitution studies with artificial vesicles [46]; thus, we have assumed an enzyme rate of 0.1 µM/hr consistent with that report. The amount of CYP27B1 in cells is also not known. "
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    PLoS ONE 01/2012; 7(1):e30773. DOI:10.1371/journal.pone.0030773 · 3.23 Impact Factor
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    • "At a molecular level, there are differences in the affinity of CYP24A1, being lower for 1,25(OH)2D2 than for 1,25(OH)2D3(56). However, 25(OH)D2 and 25(OH)D3 are metabolised at similar rates by CYP27B1(57,58) and CYP24A1 enzymes(57), although differences in the hydroxylation products and downstream metabolites of vitamin D2 and vitamin D3 may exist(59,60). At the physiological level, however, the majority of evidence suggests no difference in the suppressive effect of 25(OH)D2 and 25(OH)D3 on PTH(31,32,61–64). "
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