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  • UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1.

UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1.

Biochemical and biophysical research communications (2013-04-04)
Kaori Yasuda, Mariko Endo, Shinichi Ikushiro, Masaki Kamakura, Miho Ohta, Toshiyuki Sakaki
ABSTRACT

CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D3 or vitamin D2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D2 was produced without additional vitamin D2. Endogenous ergosterol was likely converted into vitamin D2 by UV irradiation and thermal isomerization, and then the resulting vitamin D2 was converted to 25-hydroxyvitamin D2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D2 without a substrate could be useful for practical purposes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
β-Cyclodextrin, Wacker Chemie AG, ≥95.0% (HPLC)
Sigma-Aldrich
β-Cyclodextrin, Wacker Chemie AG, ≥98.0% (HPLC)
Sigma-Aldrich
25-Hydroxyvitamin D2, ≥95.0% (HPLC)
Sigma-Aldrich
β-Cyclodextrin, ≥97%
Sigma-Aldrich
β-Cyclodextrin, powder, BioReagent, suitable for cell culture, ≥97%
Calcitriol, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
β-Cyclodextrin, Wacker Chemie AG
Supelco
1α,25-Dihydroxyvitamin D3 solution, 5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
Supelco
25-Hydroxyvitamin D2 solution, 50 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
Sigma-Aldrich
1α,25-Dihydroxyvitamin D3, ≥97.0% (HPLC)
Sigma-Aldrich
Ergosterol, ≥75%
Sigma-Aldrich
1α,25-Dihydroxyvitamin D3, ≥99% (HPLC)
Sigma-Aldrich
3-epi-25-Hydroxyvitamin D3, 98% (CP)