CONSTRUCTION OF A P450C27 FUSION ENZYME - A USEFUL TOOL TOR ANALYSIS OF VITAMIN-D-3 25-HYDROXYLASE ACTIVITY

Liver mitochondrial P450c27, encoded by the CYP27 gene, can catalyse t he 25-hydroxylation of vitamin D-3 and the 27-hydroxylation of sterols . To facilitate the study of this enzyme in cell culture systems, we e ngineered a fusion protein consisting of P450c27 coupled to its electr on-transport accessory proteins, ferredoxin and ferredoxin reductase, and assessed its enzyme activity by measuring the C-25 and C-27 (side- chain) hydroxylation of 1 alpha-hydroxyvitamin D-3 (1 alpha-OH-D-3). W hen incubated with 1 alpha-OH-D-3, COS-1 cells transfected with a vect or expressing the fusion protein produced 1 alpha,25-(OH)(2)D-3 and 1 alpha,27-(OH)(2)D-3 about four times more efficiently than did cells t ransfected with three individual components of the fusion. When incuba ted with the natural substrate, vitamin D-3, the efficiency of hydroxy lation was lower than that for 1 alpha-OH-D-3 but still 1.7-fold highe r for the fusion protein than for its individual components. The fusio n protein was also able to reproduce qualitatively and quantitatively the activity shown by P450c27 in liver cells in situ. The P450c27-ferr edoxin reductase-ferredoxin fusion construct represents a valuable too l for establishing the substrate specificity of this liver cytochrome and for evaluating its potential for activating pro-drug analogues of vitamin D.