School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
J Steroid Biochem Mol Biol. 2010 Apr;119(3-5):171-9. doi: 10.1016/j.jsbmb.2010.02.022. Epub 2010 Mar 1.
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.
CYP27B1 催化 25-羟基维生素 D3 的 1α-羟化，生成维生素 D3 的活性形式 1α,25-二羟维生素 D3。为了进一步表征小鼠 CYP27B1，将其在大肠杆菌中表达，纯化，并在磷脂囊泡中加入底物进行活性测量，囊泡模拟线粒体内膜。囊泡中的 25-羟基维生素 D3 和 25-羟基维生素 D2 以相似的动力学进行 1α-羟化，催化速率常数（kcat）分别为 41 和 48mol/min/mol P450，而 K(m) 值分别为 5.9 和 4.6mmol/mol 磷脂。当膜中的底物浓度大于 4 倍 K(m)时，CYP27B1 显示出抑制作用，与 25-羟基维生素 D3 相比，25-羟基维生素 D2 的抑制作用更为明显。用二油酰基磷脂酰胆碱和心磷脂制备的囊泡显示出比二肉豆蔻酰基磷脂酰胆碱囊泡更高的催化效率。CYP27B1 还催化囊泡相关的 24R,25-二羟维生素 D3 和 20-羟维生素 D3 的 1α-羟化，以及 1α-羟基维生素 D3 和 1α-羟基维生素 D2 的 25-羟化，但效率比 25(OH)D3 低得多。本研究表明，CYP27B1 可以催化与磷脂膜结合的 25-羟基维生素 D2 和 25-羟基维生素 D3 的羟化，这是迄今为止报道的该酶的最高活性。表达的酶在膜中 25-羟基维生素 D 浓度较高时活性较低，表明底物抑制可能有助于该酶活性的调节。
