Hosseinpour F, Hidestrand M, Ingelman-Sundberg M, Wikvall K
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Uppsala, SE-751 23, Sweden.
Biochem Biophys Res Commun. 2001 Nov 9;288(4):1059-63. doi: 10.1006/bbrc.2001.5879.
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.
猪细胞色素P450 2D25（CYP2D25），即微粒体维生素D(3) 25 -羟化酶，催化激素原维生素D(3)生物活化过程中的关键第一步。尽管CYP2D25与细胞色素P450 2D亚家族的其他成员，如人类CYP2D6，具有高度的序列同一性，但维生素D(3) 25 -羟化酶活性是细胞色素P450 2D酶中一种独特的特性。除了25 -羟化作用外，CYP2D25还可代谢药物托特罗定。在本研究中，CYP2D25在酿酒酵母W(R)菌株中实现了功能表达，并利用定点诱变技术研究底物识别位点3（SRS - 3）对CYP2D25催化特异性的作用。将CYP2D25的SRS - 3中的五个残基同时突变为CYP2D6中的等效残基，CYP2D6在25 -羟化反应中无活性。对转化酵母细胞微粒体的蛋白质免疫印迹分析表明，野生型和突变型CYP2D25的表达水平相当。重组突变型CYP2D25的25 -羟化酶活性完全丧失，而对托特罗定的活性几乎未受影响。结果表明，CYP2D25的SRS - 3中的残基是其在维生素D(3)代谢中发挥功能的重要决定因素。
