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

The Journal of Steroid Biochemistry and Molecular Biology
J L OmdahlR Rayb

Abstract

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,...Continue Reading

References

Apr 1, 1995·Endocrine Reviews·R BouillonA W Norman
Sep 1, 1996·Archives of Biochemistry and Biophysics·N Swamy, R Ray
Mar 14, 2000·Bioorganic & Medicinal Chemistry Letters·N SwamyR Ray
Jan 19, 2002·Nature Structural Biology·Christel VerbovenCamiel De Ranter
Jan 10, 2003·Journal of Cellular Biochemistry·J L OmdahlR Serda

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Citations

Dec 8, 2009·Journal of Molecular Biology·Andrew J AnnaloraC David Stout

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