Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24

Abstract

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 1α,25‐dihydroxyvitamin D 3 [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 1α,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 1α,25‐dihydroxyvitamin D 3 clearly indicated that CYP24 catalyses six‐step monooxygenation to convert 1α,25‐dihydroxyvitamin D 3 into calcitroic acid which is known as a final metabolite of 1α,25‐dihydroxyvitamin D 3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25‐hydroxyvitamin D 3 and 1α,25‐dihydroxyvitamin D 3 .