Document Detail


The importance of residues in substrate recognition site 3 for the catalytic function of CYP2D25 (vitamin D 25-hydroxylase).
MedLine Citation:
PMID:  11689019     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
F Hosseinpour; M Hidestrand; M Ingelman-Sundberg; K Wikvall
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  288     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2001 Nov 
Date Detail:
Created Date:  2001-11-05     Completed Date:  2001-12-18     Revised Date:  2012-01-30    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1059-63     Citation Subset:  IM    
Copyright Information:
Copyright 2001 Academic Press.
Affiliation:
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Uppsala, SE-751 23, Sweden.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Benzhydryl Compounds / metabolism*
Binding Sites
Blotting, Western
Catalysis
Catalytic Domain*
Cholecalciferol / metabolism
Chromatography, High Pressure Liquid
Cresols / metabolism*
Cytochrome P-450 CYP27A1
Cytochrome P-450 CYP2D6 / chemistry,  genetics,  metabolism
Microsomes / metabolism
Mutagenesis, Site-Directed*
Mutation
Phenylpropanolamine*
Saccharomyces cerevisiae / cytology,  genetics,  metabolism
Steroid Hydroxylases / chemistry*,  genetics,  metabolism*
Substrate Specificity
Swine
Grant Support
ID/Acronym/Agency:
1-R01 GM60548-01A2/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Benzhydryl Compounds; 0/Cresols; 124937-51-5/tolterodine; 14838-15-4/Phenylpropanolamine; 67-97-0/Cholecalciferol; EC 1.14.-/Cytochrome P-450 CYP27A1; EC 1.14.-/Steroid Hydroxylases; EC 1.14.13.15/CYP27A1 protein, human; EC 1.14.14.1/Cytochrome P-450 CYP2D6

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