Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1

Abstract

CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1α,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (kcat) of 9.8 min−1 and Km of 0.49 mol/mol phospholipid (510 μM phospholipid). The Km value of vitamin D3 was similar for that of cholesterol, but the kcat was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a kcat/Km that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest kcat for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.

Metabolism of 20-hydroxyvitamin D3 by mouse liver microsomes

“…Since liver mitochondria contain at least one P450, CYP27A1, that can metabolize 20(OH)D3 (19), we tested the level of contamination of the microsomal fraction with mitochondria using cytochrome c oxidase as a marker. The mitochondrial fraction exhibited a cytochrome oxidase activity of 6.75 ± 0.6 nmol/min/mg protein (data are mean ± SD for triplicate determinations), while the microsomal fraction exhibited an activity of 0.37 ± 0.05 nmol/min/mg protein.…”

“…22-Hydroxyvitamin D3 [22(OH)D3], 17,20(OH) 2 D3, 20,22(OH) 2 D3 and 20,23(OH) 2 D3 were also produced from vitamin D3 using bovine CYP11A1 (16). 20,24-dihydroxyvitamin D3 [20,24(OH) 2 D3] and 20,25(OH) 2 D3 were produced by the action of rat Cyp24a1 on 20(OH)D3 (18) while 20,26-dihydroxyvitamin D3 [20,26(OH) 2 D3] was made from 20(OH)D3 using human CYP27A1 (19). The structures of these metabolites have previously been determined by NMR (5,16,18).…”

“…We detected production of 1α-hydroxy derivatives of 20(OH)D3 and 20,23(OH) 2 D3 in vivo in human placenta and epidermal keratinocytes (8). Both CYP27A1 and CYP24A1 convert 20(OH)D3 to 20,25-dihydroxyvitamin D3 [20,25(OH) 2 D3] with this metabolite displaying greater inhibition of melanoma cell proliferation than the parent 20(OH)D3, or 1,25(OH) 2 D3 (18,19). …”

“…CYP2R1 is generally regarded as the major 25-hydroxylase in humans (20,21) although there may be some contribution from mitochondrial CYP27A1 (19,22). The knockout of both Cyp2r1 and Cyp27a1 in mice only caused a 50% decrease in serum 25-hydroxyvitamin D3 [25(OH)D3] levels indicating that in rodents there is another vitamin D 25-hydroxylase (23).…”

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

“…Recently, we reported that 20(OH)D3 can be hydroxylated by two vitamin D-metabolizing enzymes, human CYP27A1 (Tieu et al, 2012a) and rat CYP24A1 (Tieu et al, 2012b). The main products of CYP24A1 action on 20(OH)D3 are 20,24(OH) 2 D3 and 20,25(OH) 2 D3, and for CYP27A1 are 20,25(OH) 2 D3 and 20,26(OH) 2 D3.…”