CLONING, STRUCTURE, AND EXPRESSION OF A CDNA-ENCODING VITAMIN-D-3 25-HYDROXYLASE

The microsomal cytochrome P450 catalyzing the first step in the metabo lic activation of vitamin D-3 into its hormonal form 1 alpha,25-dihydr oxyvitamin D-3 has earlier been purified from pig liver. The present c ommunication describes the cloning, structure, and expression of a cDN A encoding pig liver microsomal vitamin D-3 25-hydroxylase. DNA sequen ce analysis of the cDNA revealed a 25-hydroxylase protein of 500 amino acids with a predicted molecular weight of 56,374. The structure of v itamin D-3 25-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, shows 70-80% identity with me mbers of the CYP2D family. Transfection of the vitamin D-3 25-hydroxyl ase cDNA into simian COS cells resulted in the synthesis of an enzyme that was recognized by a monoclonal antibody raised against purified v itamin D-3 25-hydroxylase and catalyzed 25-hydroxylation in the bioact ivation of vitamin D-3. Northern blot analysis showed that the mRNA fo r vitamin D-3 25-hydroxylase is found in liver and kidney. (C) 1997 Ac ademic Press.