Journal of Steroid Biochemistry & Molecular Biology 89–90 (2004) 159–162 Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue  J.L. Omdahl a , N. Swamy b , R. Serda a , A. Annalora a , M. Berne b , R. Rayb b,∗ a University of New Mexico School of Medicine, Albuquerque, NM 87131, USA b Boston University School of Medicine, Boiorganic Chemistry and Structural Biology, M Building, 10th Floor, 80 East Concord Street, Boston, MA 02118, USA Abstract 25-hydroxyvitamin D 3 - or 1,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. © 2004 Elsevier Ltd. All rights reserved. Keywords: Vitamin D cytochrome P450C24 or CYP24; Affinity labeling; Mutational analysis; Structure-function studies 1. Introduction In the Vitamin D endocrine pathway, 25-hydroxyvitamin D 3 - or 1,25-dihydroxyvitamin D 3 -24-hydroxylase (cy- tochrome P450C24, CYP24) converts 25-hydroxyvitamin D 3 (25-OH-D 3 ) to 24(R),25-dihydroxyvitamin D 3 (24, 25(OH) 2 D 3 ) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) to 1,24(R),25-trihydroxyvitamin D 3 (1,24,25(OH) 3 D 3 ) prior to their ultimate catabolic degradation (Fig. 1). Therefore, CYP24 has a dual role of removing 25-OH-D 3 from circu- lation and excess 1,25(OH) 2 D 3 from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme in the Vitamin D pathway that maintains essential tissue levels of 1,25(OH) 2 D 3 . 1,25(OH) 2 D 3 is a multi-functional hormone that regulates calcium homeostasis, immune function, cellular prolifera- tion, and differentiation and apoptotic process for cell death. Recognizing the cell-regulatory properties of 1,25(OH) 2 D 3 ,  Presented at the 12th Workshop on Vitamin D (Maastricht, The Nether- lands, 6–10 July 2003). ∗ Corresponding author. Tel.: +1-617-638-8199; fax: +1-617-638-8194. E-mail address: bapi@bu.edu (R. Rayb). extensive efforts have been directed to develop 1,25(OH) 2 D 3 and its synthetic analogs as therapeutic agents for many dis- eases including cancer. Biological actions of 1,25(OH) 2 D 3 and its analogs are mediated via Vitamin D receptor (VDR), the nuclear receptor for 1,25(OH) 2 D 3 . Therefore, VDR has been an important target for developing 1,25(OH) 2 D 3 analogs with therapeutic potential [1]. Bioavailability is an important consideration in the devel- opment of 1,25(OH) 2 D 3 -based drugs. Since CYP24 controls the catabolism of 1,25(OH) 2 D 3 and majority of its synthetic analogs, bioavailability of these compounds is also regulated by CYP24. In this token, CYP24 is an important molecular target for developing 1,25(OH) 2 D 3 -based drugs. For exam- ple, analogs could be developed that would have reduced CYP24 activity and enhanced bioavailability. Additionally an inhibitor of CYP24 would also serve the same purpose. However, realization of the above mentioned ratio- nale has been seriously challenged by the lack of any structure-functional data of CYP24. CYP24 is a typical ex- ample of mitochondrial P450c enzymes containing multiple components including. NADPH ferrodoxin-reductase and an iron–sulfur protein required for the electron-transfer pro- cess. The unique constituent in this multi-component system 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2004.03.107