title: 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-(3)H]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-[3 alpha-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-[3 alpha-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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Beckman
authors.forename: P
authors.initials: P
authors.lastname: Tadikonda
authors.forename: E
authors.initials: E
authors.lastname: Werner
authors.forename: J
authors.initials: J
authors.lastname: Prahl
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: H F
authors.initials: HF
authors.lastname: DeLuca
mesh_terms: D000818:Animals; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; C021640:calcitroic acid; D002117:Calcitriol; D002112:Calcifediol