Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

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• 作者：Annalora ; Andrew ; Bobrovnikova-Marjon ; Ekaterina ; Serda ; Rita ; Lansing ; Letitia ; Chiu ; Mark L. ; et. al.
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2004
• 出版时间：May 15, 2004
• 年：2004
• 卷：425
• 期：2
• 页码：133-146
• 全文大小：490 K

文摘

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40&ndash;50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D<sub>3sub> [1,25(OH)<sub>2sub>D<sub>3sub>] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1α- and 25-hydroxyl groups on vitamin D<sub>3sub> metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K<sub>dsub> values were obtained for 1α-vitamin D<sub>3sub> (0.06μM) and 1,25-dihydroxyvitamin D<sub>3sub> (0.05μM) whereas unmodified parental vitamin D<sub>3sub> and the non-secosteroid 25-hydroxycholesterol had lower affinities with K<sub>dsub> values of 1.3 and 1.9μM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D<sub>3sub> [24,25(OH)<sub>2sub>D<sub>3sub>] (0.43μM). Kinetic analyses of the two natural substrates 25-hyroxyvitamin D<sub>3sub> [25(OH)D<sub>3sub>] and 1,25-dihydroxyvitamin D<sub>3sub> [1,25(OH)<sub>2sub>D<sub>3sub>] revealed similar K<sub>msub> values (0.35 and 0.38μM, respectively), however, the turnover number was higher for 25(OH)D<sub>3sub> compared to 1,25(OH)<sub>2sub>D<sub>3sub> (4.2 and 1min^&minus;1, respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C<sub>23sub> oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D<sub>3sub> to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C<sub>23sub>-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C<sub>24sub> oxidation of 1,24,25-trihydroxyvitamin D<sub>3sub>. Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C<sub>23sub> and C<sub>24sub> oxidation reactions.