Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1).
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.
Tang
Edith K Y
EK
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Voo
Kimberley J Q
KJ
Nguyen
Minh N
MN
Tuckey
Robert C
RC
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism; D015652:25-Hydroxyvitamin D 2 / Q000378:metabolism; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000325:Adrenodoxin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002112:Calcifediol / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D002308:Cardiolipins / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D051336:Mitochondrial Membranes / Q000201:enzymology / Q000378:metabolism; D010316:Particle Size; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D053835:Unilamellar Liposomes / Q000737:chemistry*
C480634:20-hydroxyvitamin D3; D002308:Cardiolipins; D010713:Phosphatidylcholines; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D053835:Unilamellar Liposomes; D000325:Adrenodoxin; D015652:25-Hydroxyvitamin D 2; D015650:24,25-Dihydroxyvitamin D 3; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; C017251:1,2-oleoylphosphatidylcholine; D002112:Calcifediol; D004134:Dimyristoylphosphatidylcholine