Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25].
A new member of the cytochrome P450 (CYP) 4A subfamily (CYP4A21) was recently cloned by PCR from pig liver [Lundell, Hansson, and Wikvall (2001) J. Biol. Chem. 276, 9606-9612]. This enzyme does not catalyse omega- or (omega-1)-hydroxylation of lauric acid, the model substrate for CYP4A enzymes. Instead, CYP4A21 participates in bile acid biosynthesis in the pig. Extensive studies, primarily conducted to verify the aberrant amino acids found in CYP4A21 within a normally conserved CYP4A motif, revealed that besides CYP4A21 two additional sequences were co-amplified by PCR. These two sequences (designated CYP4A24 and CYP4A25), generated from both pig liver and kidney, were characterized by restriction-enzyme analysis and were subsequently cloned. The deduced amino acid sequences of CYP4A24 and CYP4A25 share extensive sequence identity (97%). Both enzymes, expressed in yeast cells, exhibit omega-and (omega-1)-hydroxylase activities towards lauric acid and palmitic acid. The positions of the variable regions between CYP4A24 and CYP4A25, which are confined to beta-sheets 1 and 4, indicate a possible difference in substrate specificity or regioselectivity. The porcine CYP4A21, CYP4A24 and CYP4A25 enzymes, with an overall identity of 94%, have probably evolved from a common ancestral gene, perhaps in conjunction with species-specific habits.
Lundell
Kerstin
K
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden. Kerstin.Lundell@farmbio.uu.se
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D007668:Kidney / Q000201:enzymology*; D007850:Lauric Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics; D013379:Substrate Specificity; D013552:Swine
D018076:DNA, Complementary; D007850:Lauric Acids; D011994:Recombinant Proteins; C030358:lauric acid; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C454259:cytochrome P-450 4A24 (porcine); C454260:cytochrome P-450 4A25 (porcine); C095246:taurochenodeoxycholic acid 6-alpha-hydroxylase