Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli.
The coding region of the cDNA for rat kidney calcidiol 24-hydroxylase (P450cc24), 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 P450cc24. 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.
Akiyoshi-Shibata
M
M
Biotechnology Laboratory, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Sakaki
T
T
Ohyama
Y
Y
Noshiro
M
M
Okuda
K
K
Yabusaki
Y
Y
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli; D007668:Kidney / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D015183:Restriction Mapping; D013250:Steroid Hydroxylases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D000906:Antibodies; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; D002112:Calcifediol