Human 25-Hydroxyvitamin D3-24-Hydroxylase, a Multicatalytic Enzyme

Abstract

Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-³H]D₃ substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system. These products exactly comigrated with authentic standards for 24-oxo-25-OH-D₃, 23(S),25-(OH)₂D₃, 24(R),25-(OH)₂D₃, and 24-oxo-23(S),25-(OH)₂D₃. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)₂D₃ nor any of the other 25-OH-D₃ metabolites. The products were isolated and subjected to a normal-phase amino HPLC for further separation, purification, and characterization. Comigration on two HPLC systems, periodate cleavage reactions, and NaBH₄ reduction established clearly the identity of these metabolites. Incubation of recombinant h-P450cc24 with 25-OH-[3α-³H]D₃ led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D₃. Treatment of putative 24,25,26,27-tetranor-23-OH-[3α-³H]D₃ with acetic anhydride changed its migration on amino HPLC to a less polar position, indicating acetylation of a hydroxyl group(s). These data demonstrate conclusively that h-P450cc24 is a multicatalytic enzyme catalyzing most, if not all, of the reactions in the C-24/C-23 pathway of 25-OH-D₃ metabolism. It is likely that this enzyme by itself converts 25-OH-D₃ and 1,25-(OH)₂D₃ to one of its final excretion products.