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

J. L. Omdahl, N. Swamy, Rita Serda, A. Annalora, M. Berne, R. Rayb

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)159-162
Number of pages4
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume89-90
DOIs
StatePublished - May 1 2004

Keywords

  • Affinity labeling
  • Mutational analysis
  • Structure-function studies
  • Vitamin D cytochrome P450C24 or CYP24

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Fingerprint Dive into the research topics of 'Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue'. Together they form a unique fingerprint.

Cite this