Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1).
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.
- SourceAvailable from: Andrzej Slominski[Show abstract] [Hide abstract]
ABSTRACT: Novel metabolic pathways initiated by the enzymatic action of CYP11A1 on 7DHC (7-dehydrocholesterol), ergosterol, vitamins D3 and D2 were characterized with help of chemical synthesis, UV and mass spectrometry and NMR analyses. The first pathway follows the sequence 7DHC→22(OH)7DHC → 20,22(OH)27DHC → 7DHP (7-dehydropregnenolone), which can further be metabolized by steroidogenic enzymes. The resulting 5,7-dienes can be transformed by UVB to corresponding, biologically active, secosteroids. Action of CYP11A1 on vitamin D3 and D2 produces novel hydroxyderivatives with OH added at positions C17, C20, C22, C23 and C24, some of which can be hydroxylated by CYP27B1 and/or by CYP27A1 and/ or by CYP24A1.The main products of these pathways are biologically active with a potency related to their chemical structure and the target cell type. Main products of CYP11A1-mediated metabolism on vitamin D are non-calcemic and non-toxic at relatively high doses and serve as partial agonists on the vitamin D receptor. New secosteroids are excellent candidates for therapy of fibrosing, inflammatory or hyperproliferative disorders including cancers and psoriasis.Dermato-endocrinology. 01/2013; 5(1):7-19.
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ABSTRACT: CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) and the intermediates of the C24-oxidation pathway, in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3 were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and tetranor-1,23(OH)2 D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate/mol phospholipid) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol/mol phospholipid, with 24-oxo-1,23,25(OH)3 D3 displaying the lowest and 1,24,25(OH)3 D3 displaying the highest. The kcat values varied by up to 3.8-fold with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 23-oxo-tetranor-1(OH)D3, and not tetranor-1,23(OH)2 D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as accumulation of intermediates indicating that they dissociate after each catalytic step. This article is protected by copyright. All rights reserved.FEBS Journal 06/2014; · 4.25 Impact Factor
- Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) 01/2013; 14:77-98. · 2.61 Impact Factor