Open AccessFurther Oxidation of Hydroxycalcidiol by Calcidiol 24‐Hydroxylase
Author(s) -
AkiyoshiShibata Megumi,
Sakaki Toshiyuki,
Ohyama Yoshihiko,
Noshiro Mitsuhide,
Okuda Kyuichiro,
Yabusaki Yoshiyasu
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00335.x
Subject(s) - adrenodoxin , complementary dna , recombinant dna , escherichia coli , plasmid , microbiology and biotechnology , chemistry , biochemistry , expression vector , enzyme , biology , gene , cytochrome p450
The coding region of the cDNA for rat kidney calcidiol 24‐hydroxylase ( P 450cc24), which is involved in calcium homeostasis in animals, was inserted into an expression vector pKK223‐3. The recombinant plasmid was formed in a specific manner without deletion or substitution of any parts of the coding region of the cDNA. When the resulting plasmid was introduced into Escherichia coli JM109, the recombinant cells produced a protein which was immunoreactive to an antibody against P 450cc24. When the cell‐free extract of the transformed cells was incubated with calcidiol together with bovine adrenodoxin and NADPH‐adrenodoxin reductase, not only hydroxycalcidiol but also other metabolites such as oxocalcidiol and oxohydroxycalcidiol were produced. Similarly, calcitriol was converted not only to calcitetrol but also to oxocalcitriol and oxohydroxycalcitriol. These results indicate that a single enzyme expressed in the bacteria is responsible for all these successive reactions.