The importance of residues in substrate recognition site 3 for the catalytic function of CYP2D25 (vitamin D 25-hydroxylase).

Porcine CYP2D25, microsomal vitamin D(3) 25-hydroxylase, catalyzes the essential first step in the bioactivation of the prohormone vitamin D(3). Although CYP2D25 shows a high degree of sequence identity with other members of the CYP2D subfamily, such as human CYP2D6, the vitamin D(3) 25-hydroxylase activity is a unique property among CYP2D enzymes. In addition to 25-hydroxylation, CYP2D25 also metabolizes the drug tolterodine. In this study, CYP2D25 was functionally expressed in the Saccharomyces cerevisiae W(R) strain and site-directed mutagenesis was used to study the role of substrate recognition site 3 (SRS-3) for the catalytic specificity of CYP2D25. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Western blot analysis of microsomes from transformed yeast cells showed that both the wild-type and mutant CYP2D25 were expressed at comparable levels. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained virtually unaffected. The results implicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D(3) metabolism.