Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24 -: CYP24 can convert 1α,25-dihydroxyvitamin D3 to calcitroic acid

Previously we expressed rat 25-hydroxyvitamin D-3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D-3 and 1 alpha,25-dihydroxy vitamin Dg [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 D-3 and 1 alpha,25-dihydroxyvitamin D-3, respectively. When the substrate 25-hydroxyvitamin D-3 was added to a culture of recombinant E. coli, four metabolites, 24,25-dihydroxyvitamin D-3, 24-oxo-25-hydroxyvitamin D-3, 24-oxo-23,25-dihydroxyvitamin D-3 and 24,25,26,27-tetranor-23-hydroxyvitamin D-3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D-3 Furthermore, in-vivo and in-vitro metabolic studies on 1 alpha,25-dihydroxyvitamin D-3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1 alpha,25-dihydroxyvitamin D-3 into calcitroic acid which is known as a final metabolite of 1 alpha,25-dihydroxyvitamin Dg for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D-3 and 1 alpha,25dihydroxyvitamin D-3.