title: Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells.
abstract: BACKGROUND
  The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA.
  
  
  METHODS
  We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes.
  
  
  RESULTS
  We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC(50) values of 16, 27, and 15nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC(50) of 62nM, and was sensitive to ketoconazole inhibition.
  
  
  CONCLUSIONS
  CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.
authors: Cytochroma, Inc., 100 Allstate Parkway, Suite 600, Markham, Ontario, Canada L3R 6H3.
authors: Christian
authors: C
authors: Helvig
authors: Mohammed
authors: M
authors: Taimi
authors: Don
authors: D
authors: Cameron
authors: Glenville
authors: G
authors: Jones
authors: Martin
authors: M
authors: Petkovich
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047108:Embryonic Development / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007313:Insecta / Q000821:virology; D007527:Isoenzymes; D008861:Microsomes / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072516:Retinoic Acid 4-Hydroxylase; D013379:Substrate Specificity; D014212:Tretinoin / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D009249:NADP; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C000607079:CYP26B1 protein, human; D000072516:Retinoic Acid 4-Hydroxylase