Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue.
25-hydroxyvitamin D(3)- or 1alpha,25-dihydroxyvitamin D(3)-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D(3) from circulation and excess 1,25(OH)(2)D(3) 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-D(3)-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-D(3) and 1,25(OH)(2)D(3). On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D(3) to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D(3). Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D(3) and 1,25(OH)(2)D(3)) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism.
Omdahl
J L
JL
University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Swamy
N
N
Serda
R
R
Annalora
A
A
Berne
M
M
Rayb
R
R
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D051381:Rats; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D000345:Affinity Labels; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase