Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.
Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.
Sakaki
T
T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Sawada
N
N
Nonaka
Y
Y
Ohyama
Y
Y
Inouye
K
K
D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; C021640:calcitroic acid; D002117:Calcitriol