LIVER MITOCHONDRIAL CYTOCHROME-P450 CYP27 AND RECOMBINANT-EXPRESSED HUMAN CYP27 CATALYZE 1-ALPHA-HYDROXYLATION OF 25-HYDROXYVITAMIN D-3

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D-3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D-3 and 25-hydroxylation of vitamin D-3 The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin DJ Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D-3.