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

Elaine W. Tieu, Wei Li, Jianjun Chen, Donna M. Baldisseri, Andrzej T. Slominski, Robert C. Tuckey

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 (k cat) of 9.8 min -1 and K m of 0.49 mol/mol phospholipid (510 μM phospholipid). The K m value of vitamin D3 was similar for that of cholesterol, but the k cat was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a k cat/K m 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 k cat 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.

Original languageEnglish (US)
Pages (from-to)163-171
Number of pages9
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume129
Issue number3-5
DOIs
StatePublished - Apr 1 2012

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Cholecalciferol
Metabolism
Phospholipids
Cholesterol
Cats
Calcifediol
Hydroxylation
Carbon
Mitochondrial Membranes
Membranes
Cytochrome P-450 Enzyme System
Cholesterol Side-Chain Cleavage Enzyme
20-hydroxyvitamin D3
Calcitriol
Myeloid Cells
Substrates
Catalysis
Keratinocytes
Vitamin D
Hydroxyl Radical

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Endocrinology, Diabetes and Metabolism
  • Molecular Biology
  • Cell Biology
  • Endocrinology
  • Clinical Biochemistry
  • Biochemistry

Cite this

Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1. / Tieu, Elaine W.; Li, Wei; Chen, Jianjun; Baldisseri, Donna M.; Slominski, Andrzej T.; Tuckey, Robert C.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 129, No. 3-5, 01.04.2012, p. 163-171.

Research output: Contribution to journalArticle

Tieu, Elaine W. ; Li, Wei ; Chen, Jianjun ; Baldisseri, Donna M. ; Slominski, Andrzej T. ; Tuckey, Robert C. / Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1. In: Journal of Steroid Biochemistry and Molecular Biology. 2012 ; Vol. 129, No. 3-5. pp. 163-171.
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