Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1

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Abstract

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.

Highlights

CYP27A1 metabolizes cholesterol and vitamin D3 in phospholipid vesicles. ► Comparable Km values observed for the metabolism of vitamin D3 and cholesterol. ► CYP27A1 metabolizes CYP11A1-derived 20-hydroxyvitamin D3 to two major products. ► Products identified as 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3. ► 20-Hydroxyvitamin D3 is metabolized more efficiently than vitamin D3.

Introduction

CYP27A1 is a multifunctional enzyme involved in the initial activation of vitamin D3, producing 25-hydroxyvitamin D3 (25(OH)D3), as well as in the biosynthesis of acidic and neutral bile acids. In the acidic bile acid pathway, CYP27A1 is responsible for the rate limiting step of 26-hydroxylation of cholesterol forming 26-hydroxycholesterol. Furthermore it has the ability to subsequently hydroxylate carbon 26 several times to yield 3β-hydroxy-5-cholestenoic acid [1], [2], [3]. In the neutral bile acid pathway, CYP27A1 serves to hydroxylate bile acid intermediates, 5β-cholestane-3α,7α-diol and 5β-cholestane-3α,7α,12α-triol, to initiate side chain cleavage, forming cholic acid and chenodeoxycholic acid, respectively [4]. Although primarily expressed in the liver, CYP27A1 has also been detected in keratinocytes, dermal fibroblasts, osteoblasts, arterial endothelium, parathyroid gland, ovaries and duodenum, where it could play a role in the local synthesis of 25-hydroxyvitamin D3 [5], [6], [7], [8], [9], [10].

Once formed, 25(OH)D3 is further activated by the mitochondrial 1α-hydroxylase (CYP27B1) to produce 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3. 1,25(OH)2D3 is essential for calcium and phosphorous homeostasis and thus skeletal integrity [11], [12]. In addition, 1,25(OH)2D3 has tumorostatic and anti-carcinogenic properties, where it promotes differentiation in normal and transformed cells including melanoma, leukemia, prostate, breast, keratinocytes and hematopoietic cells [13], [14]. As a result 1,25(OH)2D3 has the potential to treat hyperproliferative diseases such as psoriasis and cancer [14]. However, supraphysiological doses of 1,25(OH)2D3 are needed and this has limited its therapeutic use due to the resulting calcemic effect. As a result there is considerable interest in finding vitamin D analogs which retain the anti-proliferative property but are non-calcemic. One source of vitamin D analogs with these properties is from the metabolism of vitamin D by CYP11A1, with the major metabolite being 20-hydroxyvitamin D3 (20(OH)D3) [15], [16], [17]. This product as well as its sequential metabolites are biologically active exhibiting anti-proliferative and pro-differentiation effects on a range of cell lines including keratinocytes, leukemic and myeloid cells [18], [19], [20]. It also inhibits NF-κB activity [21] but shows no calcemic activity in rats at doses as high as 4 μg/kg [18]. Structurally similar 20(OH)D2 shows similar properties [22]. Thus, 20(OH)D3 has the potential to be used as a therapeutic drug for the treatment of hyperproliferative and inflammatory disorders. The addition of a 1α-hydroxyl group to 20(OH)D3 by CYP27B1, produces 1,20-dihydroxyvitamin D3, which exhibits moderate calcemic activity when administered at comparable doses to 20(OH)D3 [18]. However, it remains to be determined if 20(OH)D3 can undergo 25-hydroxylation by CYP27A1 or other P450s, and whether these novel products have an altered biological activity.

CYP27A1 belongs to the mitochondrial type I cytochrome P450 family, which receives its electrons from NADPH via adrenodoxin reductase and its redox partner adrenodoxin [23], [24]. CYP27A1 interacts with the matrix side of the inner mitochondrial membrane [25]. The F-G loop and the N-terminal part of the G helix have been identified as the sites of membrane attachment, similar to what has been reported for CYP24 and CYP11A1 [26], [27], [28]. As membrane bound P450s acquire their hydrophobic substrates such as vitamin D3 from the membrane phase of the phospholipid bilayer, it is important to characterize P450 activity in a membrane environment. Murtazina et al. [29] found that the activity of CYP27A1 was altered according to the presence of different phospholipid species, such as phosphatidylglycerol and phosphatidylethanolamine. However, these phospholipids are found predominantly in bacterial membranes and while they can influence the properties of the purified expressed enzyme, they are not representative of phospholipids of the inner mitochondrial membrane. Recently, unilamellar phospholipid vesicles have been used to characterize the kinetics of vitamin D metabolism by CYP11A1 and CYP27B1 [30], [31], [32]. This membrane system uses dioleoyl phosphatidylcholine and cardiolipin to closely mimic the composition of the inner mitochondrial membrane [33].

While CYP27A1 can metabolize a range of substrates including cholesterol, oxysterols and vitamin D, kinetic comparisons of the ability of CYP27A1 to metabolize different substrates are lacking. Even though one study did show that the activity of CYP27A1 towards cholesterol was about 4-fold higher than that for vitamin D3, the incubation conditions were not identical for both substrates and were not under initial rate conditions [34]. In the current study we address this deficiency by comparing the kinetic parameters for vitamin D3 and cholesterol metabolism in the phospholipid vesicle system. In addition, we describe the ability of CYP27A1 to hydroxylate the novel non-calcemic vitamin D3 analog, 20(OH)D3.

Section snippets

Materials

20(OH)D3 was enzymatically synthesized by the action of CYP11A1 on vitamin D3 and purified as described before [15]. Vitamin D3, 2-hydroxypropyl-β-cyclodextrin (cyclodextrin), NADPH, dioleoyl phosphatidylcholine, bovine heart cardiolipin and cholesterol were from Sigma-Aldrich Pty. Ltd. (Sydney, Australia). The pGro7 plasmid was from Takara Bio Inc. (Shiga, Japan). The silica gel plates were from Alugram Sil G, Macherey-Nagel, Inc. (Easton, PA). The [4-14C]cholesterol and emulsifier safe

Metabolism of cholesterol and vitamin D3 incorporated in phospholipid vesicles

Phospholipid vesicles provide a means of mimicking the inner mitochondrial membrane environment of mitochondrial P450s. Both cholesterol and vitamin D3 partition exclusively into the bilayer of phospholipid vesicles prepared in aqueous buffer [31], [42]. 25(OH)D3 has also been shown to partition greater than 97% into phospholipid vesicles [30]. As expected, the major product of vitamin D3 metabolism was identified as 25(OH)D3 based upon its identical HPLC retention time to authentic 25(OH)D3,

Discussion

In this study we have shown that purified human CYP27A1 is catalytically active towards substrates that have been incorporated into phospholipid membranes. Kinetic analysis shows that vitamin D3 metabolism by CYP27A1 has a kcat of 2.09 min−1, which is 10-fold higher than what Sawada et al. [43] reported using bacterial membranes. Our study reports the highest kcat for the 25-hydroxylation of vitamin D3 by any human cytochrome P450. Kinetic assays using membrane fractions containing CYP2R1

Acknowledgments

This work was supported by NIH [Grant R01AR052190] to AS, by the University of Western Australia and by the College of Pharmacy at the University of Tennessee Health Science Center. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

References (52)

  • N. Mast et al.

    Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1

    J. Biol. Chem.

    (2011)
  • D.A. Murtazina et al.

    Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1

    J. Lipid Res.

    (2004)
  • E.K.Y. Tang et al.

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

    J. Steroid Biochem. Mol. Biol.

    (2010)
  • R.C. Tuckey et al.

    Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

    Int. J. Biochem. Cell Biol.

    (2008)
  • R.C. Tuckey et al.

    Metabolism of 1 alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1 alpha,20-dihydroxyvitamin D3

    J. Steroid Biochem. Mol. Biol.

    (2008)
  • R.P. Gupta et al.

    Mutational analysis of CYP27A1: assessment of 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D

    Metab. Clin. Exp.

    (2007)
  • S.T. Woods et al.

    Expression of catalytically active human cytochrome P450scc in Escherichia coli and mutagenesis of isoleucine-462

    Arch. Biochem. Biophys.

    (1998)
  • J.J. Cali et al.

    Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis

    J. Biol. Chem.

    (1991)
  • R.C. Tuckey et al.

    Kinetics of the incorporation of adrenal cytochrome P-450scc into phosphatidylcholine vesicles

    J. Biol. Chem.

    (1982)
  • J. Decaprio et al.

    Bile acid and sterol solubilization in 2-hydroxypropyl-beta-cyclodextrin

    J. Lipid Res.

    (1992)
  • T. Omura et al.

    The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature

    J. Biol. Chem.

    (1964)
  • A. Hiwatashi et al.

    Purification of cytochrome P-450D1α (25-hydroxyvitamin D3-1α-hydroxylase) of bovine kidney mitochondria

    Biochem. Biophys. Res. Commun.

    (1982)
  • N. Sawada et al.

    Metabolism of vitamin D3 by human CYP27A1

    Biochem. Biophys. Res. Commun.

    (2000)
  • I. Pikuleva et al.

    Novel sterols synthesized via the CYP27A1 metabolic pathway

    Arch. Biochem. Biophys.

    (2003)
  • R. Shinkyo et al.

    Metabolism of vitamin D by human microsomal CYP2R1

    Biochem. Biophys. Res. Commun.

    (2004)
  • N. Strushkevich et al.

    Structural Analysis of CYP2R1 in Complex with Vitamin D3

    J. Mol. Biol.

    (2008)
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