Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase.
Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K(m) 9.4+/-3.3nM and V(max) 11.3+/-4.3pmolesmin(-1)pmoleP450(-1)) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.
Lutz
Justin D
JD
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195, USA.
Dixit
Vaishali
V
Yeung
Catherine K
CK
Dickmann
Leslie J
LJ
Zelter
Alex
A
Thatcher
Jayne E
JE
Nelson
Wendel L
WL
Isoherranen
Nina
N
D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D005784:Gene Amplification; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D000072516:Retinoic Acid 4-Hydroxylase; D014212:Tretinoin / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D000072516:Retinoic Acid 4-Hydroxylase