Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1.
Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1.
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DOI：
10.1016/j.jsbmb.2011.11.012
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发表时间：
2012-04
影响因子：
4.1
通讯作者：
Tuckey, Robert C
中科院分区：
文献类型：
--
作者：
Tieu, Elaine W;Li, Wei;Chen, Jianjun;Baldisseri, Donna M;Slominski, Andrzej T;Tuckey, Robert C
CYP27A1 是一种线粒体细胞色素 P450，可以分别在碳 25 和 26 处羟基化维生素 D3 和胆固醇。维生素 D3 代谢的产物 25-羟基维生素 D3，是生物活性激素 1α,25-二羟基维生素 D3 的前体。 CYP27A1 附着在线粒体内膜上，底物似乎通过膜相到达活性位点。因此，我们检查了细菌表达和纯化的 CYP27A1 代谢掺入类似于线粒体内膜的磷脂囊泡中的底物的能力。我们还检测了 CYP27A1 代谢 20-羟基维生素 D3 (20(OH)D3) 的能力，20-羟基维生素 D3 (20(OH)D3) 是一种新型非钙血症形式的维生素 D，源自 CYP11A1 对维生素 D3 的作用，维生素 D3 对角质形成细胞、白血病和骨髓细胞具有抗增殖活性。 CYP27A1 对胆固醇表现出高催化活性，周转数 (kcat) 为 9.8 min−1，Km 为 0.49 mol/mol 磷脂（510 μM 磷脂）。维生素 D3 的 Km 值与胆固醇相似，但 kcat 低 4.5 倍。 20(OH)D3 被 CYP27A1 代谢为两种主要产物，其 kcat/Km 比维生素 D3 高 2.5 倍，表明 20(OH)D3 可以有效与维生素 D3 竞争催化作用。 NMR和质谱分析表明，两种主要产物是20,25-二羟基维生素D3和20,26-二羟基维生素D3，比例几乎相等。因此，维生素 D3 侧链上 20-羟基的存在使其能够比维生素 D3 更有效地代谢，除了碳 25 之外，碳 26 也成为羟基化的主要位点。我们的研究报告了任何人类细胞色素 P450 对维生素 D3 进行 25-羟基化的最高 kcat，表明 CYP27A1 可能是 25-羟基维生素 D3 合成的重要贡献者，特别是在其高度表达的组织中。
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.