Faculty Collaboration Database

The importance of residues in substrate recognition site 3 for the catalytic function of CYP2D25 (vitamin D 25-hydroxylase). Biochem Biophys Res Commun 2001 Nov 09;288(4):1059-63

Date

11/02/2001

Pubmed ID

11689019

DOI

10.1006/bbrc.2001.5879

Scopus ID

2-s2.0-0035834602 (requires institutional sign-in at Scopus site)   14 Citations

Abstract

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.

Author List

Hosseinpour F, Hidestrand M, Ingelman-Sundberg M, Wikvall K

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Benzhydryl Compounds
Binding Sites
Blotting, Western
Catalysis
Catalytic Domain
Cholecalciferol
Cholestanetriol 26-Monooxygenase
Chromatography, High Pressure Liquid
Cresols
Cytochrome P-450 CYP2D6
Microsomes
Mutagenesis, Site-Directed
Mutation
Phenylpropanolamine
Saccharomyces cerevisiae
Steroid Hydroxylases
Substrate Specificity
Swine
Tolterodine Tartrate