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.
Hosseinpour
F
F
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Uppsala, SE-751 23, Sweden.
Hidestrand
M
M
Ingelman-Sundberg
M
M
Wikvall
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001559:Benzhydryl Compounds / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002762:Cholecalciferol / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D002851:Chromatography, High Pressure Liquid; D003408:Cresols / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010665:Phenylpropanolamine; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D000068737:Tolterodine Tartrate
D001559:Benzhydryl Compounds; D003408:Cresols; D002762:Cholecalciferol; D010665:Phenylpropanolamine; D000068737:Tolterodine Tartrate; D013250:Steroid Hydroxylases; D019389:Cytochrome P-450 CYP2D6; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase