TY - JOUR
T1 - Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue
AU - Omdahl, J. L.
AU - Swamy, N.
AU - Serda, R.
AU - Annalora, A.
AU - Berne, M.
AU - Rayb, R.
PY - 2004/5
Y1 - 2004/5
N2 - 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.
AB - 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.
KW - Affinity labeling
KW - Mutational analysis
KW - Structure-function studies
KW - Vitamin D cytochrome P450C24 or CYP24
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U2 - 10.1016/j.jsbmb.2004.03.107
DO - 10.1016/j.jsbmb.2004.03.107
M3 - Article
C2 - 15225765
AN - SCOPUS:3042601072
SN - 0960-0760
VL - 89-90
SP - 159
EP - 162
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
ER -