Construction of a P450c27 fusion enzyme

A useful tool for analysis of vitamin D3 25-hydroxylase activity

F. Jeffrey Dilworth, Stephen Matthew Black, Yu Ding Guo, Walter L. Miller, Glenville Jones

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Liver mitochondrial P450c27, encoded by the CYP27 gene,can catalyse the 25-hydroxylation of vitamin D3 and the 27-hydroxylation of sterols. To facilitate the study of this enzyme in cell culture systems, we engineered a fusion protein consisting of P450c27 coupled to its electron-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side-chain) hydroxylation of 1α-hydroxyvitamin D3 (1α-OH-D3). When incubated with 1α-OH-D3, COS-1 cells transfected with a vector expressing the fusion protein produced 1α,25-(OH)2D3 and 1α,27-(OH)2D3 about four times more efficiently than did cells transfected with three individual components of the fusion. When incubated with the natural substrate, vitamin D3, the efficiency of hydroxylation was lower than that for 1α-OH-D3 but still 1.7-fold higher for the fusion protein than for its individual components. The fusion protein was also ableto reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27-ferredoxin reductase-ferredoxin fusion construct represents a valuable tool for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.

Original languageEnglish (US)
Pages (from-to)267-271
Number of pages5
JournalBiochemical Journal
Volume320
Issue number1
DOIs
StatePublished - Nov 15 1996

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Cholestanetriol 26-Monooxygenase
Ferredoxins
Hydroxylation
Fusion reactions
Cholecalciferol
Enzymes
Liver
Oxidoreductases
Proteins
COS Cells
Prodrugs
Sterols
Cytochromes
Electron Transport
Substrate Specificity
Vitamin D
Carrier Proteins
Cell Culture Techniques
Accessories
Enzyme activity

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Construction of a P450c27 fusion enzyme : A useful tool for analysis of vitamin D3 25-hydroxylase activity. / Dilworth, F. Jeffrey; Black, Stephen Matthew; Guo, Yu Ding; Miller, Walter L.; Jones, Glenville.

In: Biochemical Journal, Vol. 320, No. 1, 15.11.1996, p. 267-271.

Research output: Contribution to journalArticle

Dilworth, F. Jeffrey ; Black, Stephen Matthew ; Guo, Yu Ding ; Miller, Walter L. ; Jones, Glenville. / Construction of a P450c27 fusion enzyme : A useful tool for analysis of vitamin D3 25-hydroxylase activity. In: Biochemical Journal. 1996 ; Vol. 320, No. 1. pp. 267-271.
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