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

• Published in: The Journal of steroid biochemistry and molecular biology 2010-04, Vol.119 (3), p.171-179
• Main Authors: Tang, Edith K.Y., Voo, Kimberley J.Q., Nguyen, Minh N., Tuckey, Robert C.
• Format: Article
• Language: English
• Subjects: 25-Hydroxyvitamin D3; Biological and medical sciences; CYP27B1; Cytochrome P450; Fundamental and applied biological sciences. Psychology; Phospholipid vesicles; Vertebrates: endocrinology; Vertebrates: reproduction; Vitamin D
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2010.02.022

CYP27B1 catalyzes the 1α-hydroxylation of 25-hydroxyvitamin D3 to 1α,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 1α-hydroxylation with similar kinetics, the catalytic rate constants ( k cat) were 41 and 48 mol/min/mol P450, respectively, while K m values were 5.9 and 4.6 mmol/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 1α-hydroxylation of vesicle-associated 24 R,25-dihydroxyvitamin D3 and 20-hydroxyvitamin D3, and 25-hydroxylation of 1α-hydroxyvitamin D3 and 1α-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.