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

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Abstract

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 (kcat) were 41 and 48 mol/min/mol P450, respectively, while Km 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 Km, 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 24R,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.

Introduction

25-Hydroxyvitamin D 1α-hydroxylase (CYP27B1) hydroxylates 25-hydroxyvitamin D3 (25(OH)D3) in the 1α-position producing 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D [1], [2]. 1,25(OH)2D3 stimulates calcium absorption, and to a lesser extent phosphorous absorption, from the small intestine [1]. 1,25(OH)2D3 also has anticarcinogenic properties, affecting proliferation, differentiation and apoptosis in cells of different lineages [1], [3], [4]. The major location of CYP27B1 is in mitochondria of the proximal renal tubule, but it is also found in a number of extra-renal sites including skin, brain, colon, prostate and breast [3], [5], [6]. This gives rise to autocrine and paracrine roles of 1,25(OH)2D3, particularly in relation to anti-proliferative and pro-differentiation actions.

Despite its importance, CYP27B1 has been poorly studied to date due to its very low concentration in kidney mitochondria and its lability once extracted with detergents [7], [8]. Hiwatashi et al. [7] reported purifying a small amount of bovine CYP27B1 to electrophoretic homogeneity. More recently, Uchida et al. [9] have reported the purification of mouse CYP27B1 expressed in E. coli.

CYP27B1 belongs to the mitochondrial (Type-1) cytochrome P450 class which receive electrons to support their hydroxylation reactions from NADPH via adrenodoxin reductase and adrenodoxin [10]. Two of the mitochondrial P450s, CYP27A1 and CYP11A1, appear to be anchored to the mitochondrial membrane primarily by a region involving the F-G loop [11], [12], [13], [14]. These P450s can hydroxylate both vitamin D and cholesterol [12], [15], [16], [17], [18], and substrates appear to reach the active site from the membrane phase [11], [13], [14], [19]. Since CYP27B1 belongs to the same family as CYP27A1, is associated with the inner mitochondrial membrane and uses relatively hydrophobic hydroxyvitamin D metabolites as substrates, it is reasonable to predict that it is also anchored to the membrane by its F-G loop region and accesses substrate from the membrane phase. Its substrate, 25(OH)D3, has been shown to partition efficiently (95%) into the membrane phase of liposomes made from egg phosphatidylcholine [20]. In the present study, we have investigated the ability of expressed mouse CYP27B1 to metabolize substrates incorporated into phospholipid vesicles. We show that 25(OH)D3 in vesicles is efficiently metabolized by CYP27B1 at low substrate concentrations, whereas at high substrate concentrations, marked substrate inhibition is observed.

Section snippets

Materials

20(OH)D3 was produced enzymatically by the action of CYP11A1 on vitamin D3 and purified by TLC and reverse-phase HPLC as described before [18]. Vitamin D3, 1,25(OH)2D2, dioleoyl phosphatidylcholine and bovine heart cardiolipin were from Sigma (St. Louis, MO). 1(OH)D2, 1(OH)D3, 25(OH)D3, 25(OH)D2 and CHAPS were from Merck (Darmstadt, Germany). 1,25(OH)2D3 and 24,25(OH)2D3 were a gift from Dr Milan Uskokovic (Hoffmann-La Roche, Nutley, NJ). [3H]25(OH)D3 and [4-14C]cholesterol were from

Expression and purification of mouse CYP27B1 and adrenodoxin

The level of expression of mouse CYP27B1 was 20 nmol/L culture. This was measured after Ni-affinity chromatography since the low level of expression and the presence of some cytochrome P420 prevented measurement of its concentration via CO-reduced minus reduced difference spectroscopy directly in the CHAPS extract of the bacterial cells. Despite using a similar procedure to Uchida et al. [9], including co-expression with chaperones GroES and GroEL, and optimizing expression time, our expression

Discussion

We obtained relatively low expression of mouse CYP27B1 compared to that reported by Uchida et al. [9] using a similar procedure. These workers reported a 10-fold increase in expression to levels as high as 300 nmol/L culture by co-expression with chaperones GroEL/ES but in our hands these chaperones only marginally increased CYP27B1 expression. Despite the low expression we were able to obtain a reasonably pure preparation of enzyme for catalytic studies from a combination of Ni affinity-,

Acknowledgements

This work was supported by the University of Western Australia Research Grants Scheme. We thank Prof. Paul Attwood for help in deriving kinetic equations to describe substrate inhibition.

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