Cloning, structure, and expression of a cDNA encoding vitamin D3 25-hydroxylase.
The microsomal cytochrome P450 catalyzing the first step in the metabolic activation of vitamin D3 into its hormonal form 1 alpha,25-dihydroxyvitamin D3 has earlier been purified from pig liver. The present communication describes the cloning, structure, and expression of a cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase. DNA sequence analysis of the cDNA revealed a 25-hydroxylase protein of 500 amino acids with a predicted molecular weight of 56,374. The structure of vitamin D3 25-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, shows 70-80% identity with members of the CYP2D family. Transfection of the vitamin D3 25-hydroxylase cDNA into simian COS cells resulted in the synthesis of an enzyme that was recognized by a monoclonal antibody raised against purified vitamin D3 25-hydroxylase and catalyzed 25-hydroxylation in the bioactivation of vitamin D3. Northern blot analysis showed that the mRNA for vitamin D3 25-hydroxylase is found in liver and kidney.
Postlind
H
H
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Axén
E
E
Bergman
T
T
Wikvall
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D053493:Cholestanetriol 26-Monooxygenase; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D013552:Swine; D014158:Transcription, Genetic
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase