Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue

25-hydroxyvitamin D₃- or 1α,25-dihydroxyvitamin D ₃-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D₃ from circulation and excess 1,25(OH) ₂D₃ from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D ₃ affinity analog, and showed that the 25-OH-D₃-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D₃ and 1,25(OH)₂D₃. On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)₂D₃ to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D₃. Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D₃ and 1,25(OH)₂D₃) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism.