A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] 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 D3 metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest Kd values were obtained for 1α-vitamin D3 (0.06μM) and 1,25-dihydroxyvitamin D3 (0.05μM) whereas unmodified parental vitamin D3 and the non-secosteroid 25-hydroxycholesterol had lower affinities with Kd values of 1.3 and 1.9μM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] (0.43μM). Kinetic analyses of the two natural substrates 25-hyroxyvitamin D3 [25(OH)D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] revealed similar Km values (0.35 and 0.38μM, respectively), however, the turnover number was higher for 25(OH)D3 compared to 1,25(OH) 2D3 (4.2 and 1min-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 C23 oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3 to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C23-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C24 oxidation of 1,24,25-trihydroxyvitamin D3. 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 C23 and C24 oxidation reactions.
ASJC Scopus subject areas
- Molecular Biology