Isolation, heterologous expression and functional characterization of a novel cytochrome P450 3A enzyme from a canine liver cDNA library.
A cDNA encoding a new member of the cytochrome P450 3A subfamily, P450 3A26, has been isolated from phenobarbital-induced canine liver. The sequence encodes a protein of 503 amino acids with 33 nucleotide differences conferring 22 amino acid substitutions when compared with the previously identified canine CYP3A12 enzyme. Nine of the amino acid differences are within the substrate recognition sites (SRSs) identified for P450 family 2, with five residue substitutions clustered within SRS-6. To facilitate heterologous expression in Escherichia coli, the N-terminus of 3A26 was modified. The expressed protein comigrated with a 3A-immunoreactive protein in dog liver microsomes with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than 3A12, which suggests that 3A26 corresponds to a previously noted but never characterized 3A enzyme in dogs. Functional characterization of 3A26 was undertaken with use of progesterone, testosterone and androstenedione as substrates. Assays of expressed 3A26 and 3A12 demonstrated that 3A26 displays low steroid hydroxylase activity. Identification of an additional canine 3A enzyme should increase our understanding of xenobiotic metabolism in this important animal model. These findings also suggest that 3A26 and 3A12 may be an interesting model system for the investigation of structure-function relationships involved in steroid metabolism catalyzed by members of the cytochrome P450 3A subfamily.
Fraser
D J
DJ
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, USA.
Feyereisen
R
R
Harlow
G R
GR
Halpert
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018076:DNA, Complementary / Q000302:isolation & purification*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D051381:Rats; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C475779:cytochrome P-450 CYP3A12 (canine); D010089:Oxidoreductases, N-Demethylating